UNIT-I

IMPURITY AND STABILITY STUDIES

Definition “Impurity”
“(1) Any component of the new drug substance which is not the chemical entity
defined as the new drug substance.
(2) Any component of the drug product which is not the chemical entity defined
as the drug substance or an excipient in the drug product.”( ICH Q6A:
Specifications)
Impurities
• During the manufacturing process, whether by chemical synthesis,
extraction, cell culture/fermentation, recovery from natural sources, or
any combination of these processes, impurities may arise.
• There is an ever increasing interest in impurities present in API’s.
• Recently, not only purity profile but also impurity profile has become
essential as per various regulatory requirements.
• Impurities are extraneous compounds that are not the drug substance
(API), but arise during the synthesis, extraction, purification, or storage of
the drug. Understanding the origin, control, and
Classification of Impurities I
• Organic impurities(process- and drug-related)
• Inorganic impurities
• Residual solvents
• Polymorphic forms
• Enantiomeric impurities
Organic impurities:
 They can arise during the manufacturing process and/or storage of the API.
 They can be identified or unidentified, volatile or non volatile
e.g.:
• Starting materials
• By-products
• Intermediates
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 • Degradation products
• Reagents, ligands and catalysts
Inorganic impurities:
 They can result from the manufacturing process, they are normally known and
identified and include
e.g.:
• Reagents, ligands, catalysts
• Heavy metals or other residual metals
• Inorganic salts
• Other materials, e.g. filter aids, charcoal….
Measurement of impurities is critical to the production of high-quality drug
substances. In addition to guidance from the local authorities of many countries,
a series of guidance developed in recent years by the Expert Working Group of
the International Conference on Harmonization of Technical Requirements for
Registration of Pharmaceuticals for Human Use, commonly known as ICH, have
been increasingly accepted by the pharmaceutical community. Impurities present
in excess of 0.1% should be identified and quantified by selective methods. It is
essential to know the structure of these impurities in the bulk drug in order to
alter the reaction condition and to reduce the quantity of impurity to an
acceptable level. Isolation, identification and quantification of impurities help us
in various ways, to obtain a pure drug substance with less toxicity and, safety in
drug therapy. Quantitative determination of these impurities could be used as a
method for the quality control and validation of drug substances. Regulatory
authorities such as US FDA, CGMP, TGA (Therapeutic Good Administration,
Australia), and MCA (Medical Control Agency, UK) insist on the impurity
profiling of drugs.
Origin of impurities
Impurities generally fall into three main categories:
• process impurities,
• degradation impurities, and
• contaminant impurities.
Additionally, enantiomers and polymorphs may be considered as impurities
under some circumstances.
• Process impurities arise during the manufacture of the drug substance.
• Degradation impurities arise during the storage of the drug substance.
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 • Contaminant impurities are not drug related but are inadvertently
introduced during processing or storage, and are not part of the
synthesis, extraction, or fermentation process.
• Impurities that cause the greatest concern are those that are toxic,
defined by the US Pharmacopeia (USP) as impurities that have
significant undesirable biological activity [50].
Process Impurities:
These include inorganic impurities, organic impurities and residual solvents.
• Organic impurities may be unwanted by-products of a chemical
synthesis.
• They may arise by many different routes, for example, by the reaction
of an intermediate with the solvent rather than the desired substrate, by
cyclization in the wrong direction.
• Organic impurities can also arise from impurities in the starting
materials, for example, traces of propylamine in butylamine may lead
to the propyl analog of the drug substance or they may be reagents used
during the reaction. Unreacted starting materials and intermediates may
also be present as impurities in the drug substances. In some cases,
enzymes are used during a chemical synthesis. These materials may be
present in the drug substance as impurities. Very frequently, batches of
drug substance made by different synthetic routes will contain different
impurities.
• In a chemical synthesis, the unwanted compounds that are not removed
during the synthetic or purification steps will become impurities. In a
similar fashion, the extraction, purification, and later synthetic steps for
natural, fermentation, or recombinant products may also give rise to
such impurities.
• Biotechnological processes may give rise to impurities such as media
components and host cell proteins. For animal and plant sources, the
manufacturing process begins with the organ, fluid, or tissue of the
animal or the whole plant or part of the plant.
• Understanding the source of the impurity will make it easier to devise a
means of eliminating the impurity, thus resulting in a drug substance of
improved quality.
• Inorganic impurities include water, salts from buffers, reagents,
ligands, catalysts, heavy metals, or other residual metals, and inorganic
compounds used in processing, such as filter aids and charcoal.
Inorganic impurities can also arise by leaching from equipment as a
result of the unit manufacturing process. Residual solvents are
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 considered a subset of organic impurities. Solvents used to create a
solution or suspension during the manufacturing process may not be
completely eliminated in the course of manufacture. Solvents used later
in the synthesis are more likely to be present in the drug substance,
although solvents that have low volatility may persist from earlier
steps.
Degradation Impurities:
 Normally, degradants are chemical breakdown compounds of the drug
substance formed during storage. In rare cases, degradants are formed when the
drug substance chemically interacts with other compounds or contaminants.
 In addition, degradants may also be formed by physical degradation, for
example, aggregates of proteinaceous material, dimmers, trimers, and so forth,
of synthetic compounds, polymorphs of synthetic compounds.
 Degradants may be chemically identical to process impurities.
 However, the levels of degradants will increase during storage, while the levels
of process impurities will remain constant. The rate of increase of degradants
resulting from storage is dependant on the chemical nature of the drug
substance.
 An understanding of the potential degradation pathways of the drug substance
will lead to optimization of the storage conditions and will result in less
impurities.
Contamination Impurities:
 Contamination impurities are unexpected adulterating compounds found in the
drug substance.
 Current manufacturing technology has reduced many of the contaminant
impurities observed in drugs prepared decades ago.
 For example, heavy metals like lead that leached from pipes or
manufacturing/storage tanks gave rise to the commonly used limit test for heavy
metals in the drug substance.
 Current pipes and tanks are primarily stainless steel or glass-lined to reduce this
concern, although the type of material is ultimately dependent on the nature of
the reactions, the nature of the drug substance, and the nature of the
manufacturing unit operations.
 Other contaminants could be, but not likely agents sprayed to improve the
environment in the manufacturing plant or accidental droppings like human hair
or paint chips from walls.
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 Disinfectants such as mono-, di-, or trichloroacetic acids or chloramines may be
present.

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Control of Drug Substance Impurities:
 In theory, all impurities should be eliminated. In practice, it is generally not
economically feasible to totally eliminate all impurities. However, the levels of
all impurities should be controlled to provide a consistent product.
 In most cases, only low levels of impurities should be allowed, but in rare cases,
even quite high levels of impurities are tolerated.
 Generally, the reaction conditions are adjusted to reduce the amounts of
byproducts produced during each step of the reaction. The reaction conditions
are tightly controlled to prevent varying levels of impurities, or even new
impurities, from arising.
 High-quality starting materials may also lead to lower amounts of impurities in
the final product when starting material impurities are carried through to drug
substance impurities.
Similarly, the use of high-quality reagents may help avoid the generation
of unwanted by-products. Other options to reduce these impurities are the
introduction of additional intermediate or final purification steps.
Draft active drug substance guidance published back in June 1998
provide, draft recommendations for including information in abbreviated new
drug applications (ANDAs) and supporting drug master files (DMFs) on the
identification and qualification of impurities in drug substances produced by
chemical syntheses for both monograph and non-monograph drug substances.
Impurities in drug substances are addressed from two perspectives:
 CHEMISTRY ASPECTS including classification and identification of
impurities, generating analytical reports, setting specifications, and a brief
discussion of analytical procedures
 SAFETY ASPECTS including comparative studies and genotoxocity
testing.

Evaluate & Compare Innovator Drug Impurities

Specific guidance is provided for:

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  Qualifying impurities found in the drug substance used for the ANDA
via a comparison with impurities found in the related USP
monograph, scientific literature, or innovator material.
 Qualifying impurities found at higher levels in the drug substance
used for the ANDA than found in the related USP monograph,
scientific literature, or innovator material;
 Qualifying impurities in the drug substance used for the ANDA that
are not found in the related USP monograph, scientific literature, or
innovator material;
The June 1998 FDA draft guidance is not applicable to the following
classes:-
• biological and biotechnological
• peptides,
• oligonucleotides
• radio-pharmaceuticals
• fermentation & derived semi-synthetic
• products
• herbal products
• crude products of animal/plant origin.
10 'RULES TO REMEMBER'
1. Rule No.1 - Evaluate the RLD impurity profile (i.e. get a baseline).
2. Rule No.2. Treat with CAUTION or REJECT a vendor profile
HIGHER than the innovator material.
3. Rule No.3. LOOK at impurity profiles in the major pharmacopoeia
(USP / BP / JP) and compare with vendor's dedicated synthesis
(comparing profiles is important)
4. Rule No.4. 'Approved vendors‘ may have unique impurities due to the
purifying process. LOOK for these 'specified impurities' in the actives
chromatograms (i.e. "Stress the Active material").
5. Rule No.5. Unknown impurities must not exceed 0.1% (if they do, go
back to active vendor to clean up material).
6. Rule No.6. Organic impurities are the main focus in impuritiy profiles
(Note: residual solvents have there own guideline and limits).
7. Rule No.7. Do get the DMF holder to state the 'specific impurities' and
the potential impurities (i.e. those impurities which do arise and those
which

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 8. Rule No.8. Always stress the active in-house to see which impurities do
occur.
9. Rule No.9. In drug development, if the active has an unknown >0.1% -
and it can not be reduced - Look for an alternative supply with a better
profile.
10. Rule No.10. REMEMBER an unknown impurity close to 0.1% may
grow to >0.1% on stability (ageing). There's no such concept as a safe
unknown >0.1% potential impurities
Structural analysis data and information which of the analytical methods
described have been used to detect that Impurity.

Discussion of possible routes of degradation

 Analytical methods (LOD and LOQ) used to detect likely impurities or
other related impurities

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IMPURITIES IN NEW DRUG PRODUCTS:
Guidance for Industry Q3B(R2) Impurities in New Drug Products:
• This guidance provides recommendations for registration applications on
the content and qualification of impurities in new drug products produced
from chemically synthesized new drug substances not previously
registered in a region or member state.
• This guidance revises the ICH guidance of the same title that was issued
in May 1997 and first revised in February 2003.
• The first revision clarified the 1997 guidance and included other
changes.3 The revision also provided consistency with more recently
published ICH guidances (e.g., Q3A(R) Impurities in New Drug
Substances, Q3C Impurities: Residual Solvents, and Q6A Specifications:
Test Procedures and Acceptance Criteria for New Drug Substances and
New Drug Products: Chemical Substances).
• This second revision provides clarification to Attachment 2.4 This
guidance complements the ICH Q3A(R) guidance, which should be
consulted for basic principles along with ICH Q3C when appropriate.
RATIONALE FOR THE REPORTING AND CONTROL OF DEGRADATION
PRODUCTS
• The applicant should summarize the degradation products observed
during manufacture and/or stability studies of a new drug product.
• This summary should be based on sound scientific appraisal of potential
degradation pathways in the new drug product and impurities arising from
the interaction with excipients and/or the immediate container closure
system.
• In addition, the applicant should summarize any laboratory studies
conducted to detect degradation products in the new drug product. This
summary also should include test results of batches manufactured during
the development process and batches representative of the proposed
commercial process.
• A rationale should be provided for exclusion of those impurities that are
not degradation products (e.g., process impurities from the drug substance
and impurities arising from excipients).
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 • The impurity profiles of the batches representative of the proposed
commercial process should be compared with the profiles of batches used
in development, and any differences should be discussed.
• Any degradation product observed in stability studies conducted at the
recommended storage condition should be identified when present at a
level greater than (>) the identification thresholds given in Attachment 1.
When identification of a degradation product is infeasible, a summary of
the laboratory studies demonstrating the unsuccessful efforts to identify it
should be included in the registration application.
• Degradation products present at a level of not more than (≤) the
identification threshold generally would not need to be identified.
However, analytical procedures should be developed for those degradation
products that are suspected to be unusually potent, producing toxic or
significant pharmacological effects at levels not more than (≤) the
identification threshold.
• In unusual circumstances, technical factors (e.g., manufacturing
capability, a low drug substance to excipient ratio, or the use of excipients
that are crude products of animal or plant origin) can be considered part of
the justification for selection of alternative thresholds based on
manufacturing experience with the proposed commercial process.
Specification of Impurities in the API
• Impurities Testing Guideline:
• Impurities in New Drug Substances (ICH Q3A (R2))
• Specific pharmacopoeial monograph and general monograph “Substances for
pharmaceutical use”
Use of Ph.Eur. Monographs for API
• Specific monograph,
e.g. Gentamicine sulphate, Acetazolamide
General texts:
e.g. residual solvents
impurities in substances for pharmaceutical use
General monographs
– Products of fermentation
– Products with a risk of transfer of spongiform encephalopathies of animal
origin
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 – Substances for pharmaceutical use
Impurities originating from degradation of the drug substance:
• Impurities can also be formed by degradation of the end product during
manufacturing of bulk drugs.
• Degradation products resulting from storage or formulation to different
dosage forms or aging are common impurities in the medicines.
• The definition of degradation product in the ICH guidelines is a molecule
resulting from a chemical change in the substance brought about by
overtime or due to the action of light, temperature, pH or water or by
reaction with excipient and/or the intermediate container closure system .
• For example in the case of aspartame, in the presence of moisture,
hydrolysis occurs to form the degradation products L- aspartyl-
Lphenyalanine and 3-benzyl-6-carboxymethyl 2, 5-diketopierazine. A
third degradation product β-L- aspartyl-L-phenylalanine methyl ester is
also known to form. Aspartame degradation also occurs during prolonged
heat treatment.
ICH guidelines:
• According to ICH guidelines, each impurity must be investigated with
respect to both chemistry and safety aspects.
• The former include identification (structural characterization), reporting
and quantitation using suitable analytical procedures, while the latter
include a process of acquiring and evaluating data concerning the
biological safety of an impurity (qualification).
• Individually listed impurities, limited with specific acceptance criteria,
are referred to as specified and they can be either identified or
unidentified.
• Unspecified impurities are limited by a general acceptance criterion. A
decision tree for the identification and qualification along with the
corresponding thresholds, which are dependent on the maximum
permitted daily dose (MDD), is given by ICH.
• Summing up, the following list of organic impurities must be presented
in the specification of a synthetic drug substance:
• - Each specified identified or unidentified impurity
• - Any unspecified impurity
• - Total impurity
Specified unidentified impurities are referred to by an appropriate qualitative
analytical description (e.g. relative retention time).The ICH topics, codes of
quality guidelines are given in Table.
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• dose and total daily intake (TDI) of the impurities. Table1.3 provides the
ICH threshold for control of the organic impurities in new drug
substances.
• Depending on whether the maximum daily dose is higher or lower than
2g, organic impurities in a new drug substance at (or greater than) 0.05%
or 0.1% requires identification.
• Based on the maximum daily dose, the identification thresholds for
organic impurities in new drug products are divided into four groups
to give more consideration to low dose drug products.

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 • For most of the new drug products, the maximum daily dose is
between 10mg–2g/day. Therefore, any impurities at 0.2% or greater
would have to be identified.

REPORTING DEGRADATION PRODUCTS, CONTENT OF BATCHES (4.0)

• Analytical results should be provided in the registration application for all
relevant batches of the new drug product used for clinical, safety, and
stability testing, as well as batches that are representative of the proposed
commercial process.
• Quantitative results should be presented numerically, and not in general
terms such as “complies”, “meets limit.”
• Any degradation product at a level greater than (>) the reporting
threshold (see Attachment 1), and total degradation products observed in
the relevant batches of the new drug product, should be reported with the
analytical procedures indicated. Below 1.0 percent, the results should be
reported to the number of decimal places (e.g., 0.06 percent) in the
applicable reporting threshold; at and above 1.0 percent, the results
should be reported to one decimal place (e.g., 1.3 percent). Results should
be rounded using conventional rules (see Attachment 2). A tabulation
(e.g., spreadsheet) of the data is recommended.
• Degradation products should be designated by code number or by an
appropriate descriptor (e.g., retention time).
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• If a higher reporting threshold is proposed, it should be fully justified. All
degradation products at a level greater than (>) the reporting threshold
should be summed and reported as total degradation products.
• Chromatograms with peaks labeled (or equivalent data if other analytical
procedures are used) from representative batches, including
chromatograms from analytical procedure validation studies and from
long-term and accelerated stability studies, should be provided.
• The applicant should ensure that complete degradation product profiles
(e.g., chromatograms) of individual batches are available, if requested.
• For each batch of the new drug product described in the registration
application, the documentation should include:
• Batch identity, strength, and size
• Date of manufacture
• Site of manufacture
• Manufacturing process
• Immediate container closure
• Degradation product content, individual and total
• Use of batch (e.g., clinical studies, stability studies)
• Reference to analytical procedure used
• Batch number of the drug substance used in the new drug product
• Storage conditions for stability studies
LISTING OF DEGRADATION PRODUCTS IN SPECIFICATIONS
(5.0)
• The specification for a new drug product should include a list of
degradation products expected to occur during manufacture of the
commercial product and under recommended storage conditions.
• Stability studies, knowledge of degradation pathways, product
development studies, and laboratory studies should be used to characterize
the degradation profile.
• The selection of degradation products in the new drug product
specification should be based on the degradation
• products found in batches manufactured by the proposed commercial
process. Those individual
• degradation products with specific acceptance criteria included in the
specification for the new
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• drug product are referred to as specified degradation products in this
guidance.
• Specified degradation products can be identified or unidentified. A
rationale for the inclusion or exclusion of degradation products in the
specification should be presented. This rationale should include a
discussion of the degradation profiles observed in the safety and clinical
development batches and in stability studies, together with a consideration
of the degradation profile of batches manufactured by the proposed
commercial process.
• Specified identified degradation products should be included along with
specified unidentified degradation products estimated to be present at a
level greater than (>) the identification threshold given in Attachment 1.
• For degradation products known to be unusually potent or to produce
toxic or unexpected pharmacological effects, the quantitation or detection
limit of the analytical procedures should be commensurate with the level
at which the degradation products should be controlled.
• For unidentified degradation products, the procedure used and
assumptions made in establishing the level of the degradation product
should be clearly stated.
• Specified unidentified degradation products should be referred to by an
appropriate qualitative analytical descriptive label (e.g., unidentified A,
unidentified with relative retention of 0.9). A general acceptance criterion
of not more than (≤) the identification threshold (Attachment 1) for any
unspecified degradation product and an acceptance criterion for total
degradation products should also be included.
• For a given degradation product, its acceptance criterion should be
established by taking into account its acceptance criterion in the drug
substance (if applicable), its qualified level, its increase during stability
studies, and the proposed shelf life and recommended storage conditions
for the new drug product.
• Furthermore, each acceptance criterion should be set no higher than the
qualified level of the given degradation product.
• Where there is no safety concern, degradation product acceptance criteria
should be based on data generated from batches of the new drug product
manufactured by the proposed commercial process, allowing sufficient
latitude to deal with normal manufacturing and analytical variation and
the stability characteristics of the new drug product.
• Although normal manufacturing variations are expected, significant
variation in batch-to-batch degradation product levels can indicate that the
manufacturing process of the new drug product is not adequately
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 controlled and validated (see ICH Q6A guidance on specifications,
decision tree #2, for establishing an acceptance criterion for a specified
degradation product in a new drug product).
• In this guidance, the use of two decimal places for thresholds (see
Attachment 1) does not necessarily indicate the precision of the
acceptance criteria for specified degradation products and total
degradation products.
• In summary, the new drug product specification should include, where
applicable, the following list of degradation products:
• Each specified identified degradation product
• Each specified unidentified degradation product
• Any unspecified degradation product with an acceptance criterion of not more
than
• (≤) the identification threshold
• Total degradation products
QUALIFICATION OF DEGRADATION PRODUCTS (6.0)
• Qualification is the process of acquiring and evaluating data that
establishes the biological safety of an individual degradation product or a
given degradation profile at the levels specified.
• The applicant should provide a rationale for establishing degradation
product acceptance criteria that includes safety considerations.
• The level of any degradation product present in a new drug product that
has been adequately tested in safety and/or clinical studies would be
considered qualified.
• Therefore, it is useful to include any available information on the actual
content of degradation products in the relevant batches at the time of use
in safety and/or clinical studies.
• Degradation products that are also significant metabolites present in
animal and/or human studies are generally considered qualified.
• Degradation products could be considered qualified at levels higher than
those administered in safety studies based on a comparison between actual
doses given in the safety studies and the intended dose of the new drug
product.
• Justification of such higher levels should include consideration of factors
such as:

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 • (1) the amount of degradation product administered in previous safety
and/or clinical studies and found to be safe;
• (2) the increase in the amount of the degradation product; and
• (3) other safety factors, as appropriate.
• If the qualification thresholds given in Attachment 1 are exceeded and
data are unavailable to qualify the proposed acceptance criterion of a
degradation product, additional studies to obtain such data may be
appropriate (see Attachment 3).
• Higher or lower thresholds for qualification of degradation products may
be appropriate for some individual new drug products based on scientific
rationale and level of concern, including drug class effects and clinical
experience.
• For example, qualification can be especially important when there is
evidence that such degradation products in certain new drug products or
therapeutic classes have previously been associated with adverse reactions
in patients.
• In these instances, a lower qualification threshold may be appropriate.
• Conversely, a higher qualification threshold may be appropriate for
individual new drug products when the level of concern for safety is less
than usual based on similar considerations (e.g., patient population, drug
class effects, and clinical considerations).
• Proposals for alternative thresholds would be considered on a case-by-
case basis.
• The Decision Tree for Identification and Qualification of a Degradation
Product (Attachment 3) describes considerations for the qualification of
degradation products when thresholds are exceeded.
• In some cases, reducing the level of degradation product (e.g., use of a
more protective container closure or modified storage conditions) to not
more than (≤) the threshold can be simpler than providing safety data.
• Alternatively, adequate data could be available in the scientific literature
to qualify a degradation product.
• If neither is the case, additional safety testing should be considered.
• The studies considered appropriate to qualify a degradation product will
depend on a number of factors, including the patient population, daily
dose, and route and duration of new drug product administration.
Such studies can be conducted on the new drug product or substance containing
the degradation products to be controlled, although studies using isolated
degradation products can sometimes be appropriate.
GLOSSARY
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 Degradation Product: An impurity resulting from a chemical change in
the drug substance brought about during manufacture and/or storage of
the new drug product by the effect of, fo example, light, temperature, pH,
water, or by reaction with an excipient and/or the immediate container
closure system
 Degradation Profile: A description of the degradation products
observed in the drug substance or drug product
 Development Studies: Studies conducted to scale-up, optimize, and
validate the manufacturing process for a drug product
 Identification Threshold: A limit above (>) which a degradation
product should be identified
 Identified Degradation Product: A degradation product for which a
structural characterization
has been achieved.
 Impurity: Any component of the new drug product that is not the drug
substance or an excipient in the drug product
 Impurity Profile: A description of the identified and unidentified
impurities present in a drug product
 New Drug Substance: The designated therapeutic moiety that has not
been previously registered in a region or member state (also referred to as
a new molecular entity or new chemical entity). It can be a complex, simple
ester, or salt of a previously approved substance.
• Qualification: The process of acquiring and evaluating data that
establishes the biological safety of an individual degradation product
or a given degradation profile at the levels specified
• Qualification Threshold: A limit above (>) which a degradation
product should be qualified
• Reporting Threshold: A limit above (>) which a degradation
product should be reported
• Specified Degradation Product: A degradation product that is
individually listed and limited with a specific acceptance criterion in
the new drug product specification. A specified degradation product
can be either identified or unidentified
• Unidentified Degradation Product: A degradation product for
which a structural characterization has not been achieved and that is
defined solely by qualitative analytical properties (e.g.,
chromatographic retention time)

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  Unspecified Degradation Product: A degradation product that is
limited by a general acceptance criterion, but not individually listed
with its own specific acceptance criterion, in the new drug product
specification.

IMPURITIES IN RESIDUAL SOLVENTS

IMPURITIES: GUIDELINE FOR RESIDUAL SOLVENTS
INTRODUCTION
The objective of this guideline is to recommend acceptable amounts for
residual solvents in pharmaceuticals for the safety of the patient. The guideline
recommends use of less toxic solvents and describes levels considered to be
toxicologically acceptable for some residual solvents.
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 Residual solvents in pharmaceuticals are defined here as organic volatile
chemicals that are used or produced in the manufacture of drug substances or
excipients, or in the preparation of drug products. The solvents are not
completely removed by practical manufacturing techniques. Appropriate
selection of the solvent for the synthesis of drug substance may enhance the
yield, or determine characteristics such as crystal form, purity, and solubility.
Therefore, the solvent may sometimes be a critical parameter in the synthetic
process. This guideline does not address solvents deliberately used as excipients
nor does it address solvates. However, the content of solvents in such products
should be evaluated and justified.
Since there is no therapeutic benefit from residual solvents, all residual
solvents should be removed to the extent possible to meet product specifications,
good manufacturing practices, or other quality-based requirements. Drug
products should contain no higher levels of residual solvents than can be
supported by safety data.
Some solvents that are known to cause unacceptable toxicities (Class 1,
Table 1) should be avoided in the production of drug substances, excipients, or
drug products unless their use can be strongly justified in a risk-benefit
assessment. Some solvents associated with less severe toxicity (Class 2, Table 2)
should be limited in order to protect patients from potential adverse effects.
Ideally, less toxic solvents (Class 3, Table 3) should be used where practical.
The lists are not exhaustive and other solvents can be used and later added
to the lists. Recommended limits of Class 1 and 2 solvents or classification of
solvents may change as new safety data becomes available. Supporting safety
data in a marketing application for a new drug product containing a new solvent
may be based on concepts in this guideline or the concept of qualification of
impurities as expressed in the guideline for drug substance (Q3A, Impurities in
New Drug Substances) or drug product (Q3B, Impurities in New Drug Products),
or all three guidelines.
SCOPE OF THE GUIDELINE
Residual solvents in drug substances, excipients, and in drug products are
within the scope of this guideline. Therefore, testing should be performed for
residual solvents when production or purification processes are known to result
in the presence of such solvents. It is only necessary to test for solvents that are
used or produced in the manufacture or purification of drug substances,
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excipients, or drug product. Although may choose to test the drug product, a
cumulative method may be used to calculate the residual solvent levels in the
drug product from the levels in the ingredients used to produce the drug product.
If the calculation results in a level equal to or below that recommended in this
guideline, no testing of the drug product for residual solvents need be considered.
If, however, the calculated level is above the recommended level, the drug
product should be tested to ascertain whether the formulation process has
reduced the relevant solvent level to within the acceptable amount. Drug product
should also be tested if a solvent is used during its manufacture.
This guideline does not apply to potential new drug substances, excipients,
or drug
products used during the clinical research stages of development, nor does it
apply to
existing marketed drug products.
The guideline applies to all dosage forms and routes of administration.
Higher levels of residual solvents may be acceptable in certain cases such as
short term (30 days or less) or topical application. Justification for these levels
should be made on a case by case basis.
GENERAL PRINCIPLES
Classification of Residual Solvents by Risk Assessment
The term "tolerable daily intake" (TDI) is used by the International
Program on Chemical Safety (IPCS) to describe exposure limits of toxic
chemicals and "acceptable daily intake" (ADI) is used by the World Health
Organization (WHO) and other national and international health authorities and
institutes. The new term "permitted daily exposure" (PDE) is defined in the
present guideline as a pharmaceutically acceptable intake of residual solvents to
avoid confusion of differing values for ADI's of the same substance.
Residual solvents assessed in this guideline were evaluated for their
possible risk to human health and placed into one of three classes as follows:
Class 1 solvents: Solvents to be avoided
Known human carcinogens, strongly suspected human carcinogens, and
environmental hazards.
Class 2 solvents: Solvents to be limited
23
 Non-genotoxic animal carcinogens or possible causative agents of other
irreversible toxicity such as neurotoxicity or teratogenicity. Solvents suspected of
other significant but reversible toxicities.
Class 3 solvents: Solvents with low toxic potential
Solvents with low toxic potential to man; no health-based exposure limit is
needed. Class 3 solvents have PDEs of 50 mg or more per day.
Methods for Establishing Exposure Limits
The method used to establish permitted daily exposures for residual
solvents is presented in Appendix 3. Summaries of the toxicity data that were
used to establish limits are published in Pharmeuropa, Vol. 9, No. 1, Supplement,
April 1997.
Options for Describing Limits of Class 2 Solvents
Two options are available when setting limits for Class 2 solvents.
Option 1: The concentration limits in ppm stated in Table 2 can be used. They
were calculated using equation (1) below by assuming a product mass of 10 g
administered daily.
Concentration (ppm) 1000 x PDE
Dose (1)
Here, PDE is given in terms of mg/day and dose is given in g/day.
These limits are considered acceptable for all substances, excipients, or
products.
Therefore this option may be applied if the daily dose is not known or
fixed. If all excipients and drug substances in a formulation meet the limits given
in Option 1, then these components may be used in any proportion. No further
calculation is necessary provided the daily dose does not exceed 10 g. Products
that are administered in doses greater than 10 g per day should be considered
under Option 2.
Option 2: It is not considered necessary for each component of the drug product
to comply with the limits given in Option 1. The PDE in terms of mg/day as
stated in Table 2 can be used with the known maximum daily dose and equation
(1) above to determine the concentration of residual solvent allowed in drug
product. Such limits are considered acceptable provided that it has been
24
demonstrated that the residual solvent has been reduced to the practical
minimum. The limits should be realistic in relation to analytical precision,
manufacturing capability, reasonable variation in the manufacturing process, and
the limits should reflect contemporary manufacturing standards.
Option 2 may be applied by adding the amounts of a residual solvent
present in each of the components of the drug product. The sum of the amounts
of solvent per day should be less than that given by the PDE.
Consider an example of the use of Option 1 and Option 2 applied to
acetonitrile in a drug product. The permitted daily exposure to acetonitrile is 4.1
mg per day; thus, the Option 1 limit is 410 ppm. The maximum administered
daily mass of a drug product is 5.0 g, and the drug product contains two
excipients. The composition of the drug product and the calculated maximum
content of residual acetonitrile are given in the following table.
Component Amount in Acetonitrile Daily exposure
formulation content
Drug substance 0.3 g 800 ppm 0.24 mg
Excipient 1 0.9 g 400 ppm 0.36 mg
Excipient 2 3.8 g 800 ppm 3.04 mg
Drug Product 5.0 g 728 ppm 3.64 mg

Excipient 1 meets the Option 1 limit, but the drug substance, excipient 2,
and drug product do not meet the Option 1 limit. Nevertheless, the product meets
the Option 2 limit of 4.1 mg per day and thus conforms to the recommendations
in this guideline.
Consider another example using acetonitrile as residual solvent. The
maximum administered daily mass of a drug product is 5.0 g, and the drug
product contains two excipients. The composition of the drug product and the
calculated maximum content of residual acetonitrile are given in the following
table.
Component Amount in Acetonitrile Daily exposure
formulation content
Drug substance 0.3 g 800 ppm 0.24 mg
Excipient 1 0.9 g 2000 ppm 1.80 mg
Excipient 2 3.8 g 800 ppm 3.04 mg
Drug Product 5.0 g 1016 ppm 5.08 mg
25
 In this example, the product meets neither the Option 1 nor the Option 2
limit according to this summation. The manufacturer could test the drug product
to determine if the formulation process reduced the level of acetonitrile. If the
level of acetonitrile was not reduced during formulation to the allowed limit, then
the manufacturer of the drug product should take other steps to reduce the
amount of acetonitrile in the drug product. If all of these steps fail to reduce the
level of residual solvent, in exceptional cases the manufacturer could provide a
summary of efforts made to reduce the solvent level to meet the guideline value,
and provide a risk-benefit analysis to support allowing the product to be utilised
with residual solvent at a higher level.
Analytical Procedures
Residual solvents are typically determined using chromatographic
techniques such as gas chromatography. Any harmonised procedures for
determining levels of residual solvents as described in the pharmacopoeias
should be used, if feasible. Otherwise, manufacturers would be free to select the
most appropriate validated analytical procedure for a particular application. If
only Class 3 solvents are present, a nonspecific method such as loss on drying
may be used.
Validation of methods for residual solvents should conform to ICH
guidelines Text on Validation of Analytical Procedures and Extension of the ICH
Text on Validation of Analytical Procedures.
Reporting levels of residual solvents
Manufacturers of pharmaceutical products need certain information about
the content of residual solvents in excipients or drug substances in order to meet
the criteria of this guideline. The following statements are given as acceptable
examples of the information that could be provided from a supplier of excipients
or drug substances to a pharmaceutical manufacturer. The supplier might choose
one of the following as appropriate:
 Only Class 3 solvents are likely to be present. Loss on drying is less than
0.5%.
 Only Class 2 solvents X, Y, ... are likely to be present. All are below the
Option 1
limit. (Here the supplier would name the Class 2 solvents represented by X, Y,
...)
26
  Only Class 2 solvents X, Y, ... and Class 3 solvents are likely to be present.
Residual Class 2 solvents are below the Option 1 limit and residual Class 3
solvents are below 0.5%.
If Class 1 solvents are likely to be present, they should be identified and
quantified. "Likely to be present" refers to the solvent used in the final
manufacturing step and to solvents that are used in earlier manufacturing steps
and not removed consistently by a validated process.
If solvents of Class 2 or Class 3 are present at greater than their Option 1 limits
or 0.5%, respectively, they should be identified and quantified.
LIMITS OF RESIDUAL SOLVENTS
Solvents to Be Avoided
Solvents in Class 1 should not be employed in the manufacture of drug
substances, excipients, and drug products because of their unacceptable toxicity
or their deleterious environmental effect. However, if their use is unavoidable in
order to produce a drug product with a significant therapeutic advance, then their
levels should be restricted as shown in Table 1, unless otherwise justified. 1,1,1-
Trichloroethane is included in Table 1 because it is an environmental hazard. The
stated limit of 1500 ppm is based on a review of the safety data.
TABLE 1. Class 1 solvents in pharmaceutical products (solvents that should
be avoided).
Solvent Concentration limit Concern
(ppm)

Benzene 2 Carcinogen
Carbon tetrachloride 4 Toxic and
environmental

1,2-Dichloroethane 5 Toxic
1,1-Dichloroethene 8 Toxic
1,1,1- 1500 Environmental
Trichloroethane hazard

Solvents to Be Limited

27
 Solvents in Table 2 should be limited in pharmaceutical products because
of their inherent toxicity. PDEs are given to the nearest 0.1 mg/day, and
concentrations are given to the nearest 10 ppm. The stated values do not reflect
the necessary analytical precision of determination. Precision should be
determined as part of the validation of the method.
TABLE 2. Class 2 solvents in pharmaceutical products.
Solvent PDE (mg/day) Concentration limit
(ppm)
Acetonitrile 4.1 410
Chlorobenzene 3.6 360
Chloroform 0.6 60
Cumene1 0.7 70
Cyclohexane 38.8 3880
1,2-Dichloroethene 18.7 1870
Dichloromethane 6.0 600
1,2-Dimethoxyethane 1.0 100
N,N- 10.9 1090
Dimethylacetamide
N,N- 8.8 880
Dimethylformamide
1,4-Dioxane 3.8 380
2-Ethoxyethanol 1.6 160
Ethyleneglycol 6.2 620
Formamide 2.2 220
Hexane 2.9 290
Methanol 30.0 3000
2-Methoxyethanol 0.5 50
Methylbutyl ketone 0.5 50
Methylcyclohexane 11.8 1180
Methylisobutylketone2 45 4500
N-Methylpyrrolidone3 5.3 530
Nitromethane 0.5 50
Pyridine 2.0 200
Sulfolane 1.6 160
Tetrahydrofuran4 7.2 720
Tetralin 1.0 100
Toluene 8.9 890
1,1,2-Trichloroethene 0.8 80
28
Xylene* 21.7 2170

1
The information included for Cumene reflects that included in the
Revision of PDE Information for Cumene which reached Step 4 in February
2011 and was subsequently incorporated into the core Guideline.
2
The information included for Methylisobutylketone reflects that included
in the Revision of PDE Information for Methylisobutylketone which reached
Step 4 in November 2016 and was subsequently incorporated into the core
Guideline.
3
The information included for N-Methylpyrrolidone reflects that included
in the Revision of PDE Information for NMP which reached Step 4 in September
2002 (two mistyping corrections made in October 2002), and was incorporated
into the core guideline in November 2005.
*usually 60% m-xylene, 14% p-xylene, 9% o-xylene with 17% ethyl
benzene
Solvents with Low Toxic Potential
Solvents in Class 3 (shown in Table 3) may be regarded as less toxic and of
lower risk to human health. Class 3 includes no solvent known as a human health
hazard at levels normally accepted in pharmaceuticals. However, there are no
long-term toxicity or carcinogenicity studies for many of the solvents in Class 3.
Available data indicate that they are less toxic in acute or short-term studies and
negative in genotoxicity studies. It is considered that amounts of these residual
solvents of 50 mg per day or less (corresponding to 5000 ppm or 0.5% under
Option 1) would be acceptable without justification. Higher amounts may also be
acceptable provided they are realistic in relation to manufacturing capability and
good manufacturing
practice.
TABLE 3. Class 3 solvents which should be limited by GMP or other
quality-based requirements.
Acetic acid Heptane
Acetone Isobutyl acetate
Anisole Isopropyl acetate
1-Butanol Methyl acetate
2-Butanol 3-Methyl-1-butanol
29
Butyl acetate Methylethyl ketone
tert-Butylmethyl 2-Methyl-1-propanol
ether
Dimethyl sulfoxide Pentane
Ethanol 1-Pentanol
Ethyl acetate 1-Propanol
Ethyl ether 2-Propanol
Ethyl formate Propyl acetate
Formic acid Triethylamine5

4
The information included for Tetrahydrofuran reflects that included in the
Revision of PDE Information for THF which reached Step 4 in September 2002,
and was incorporated into the core guideline in November 2005. See Part II
(pages 18-19).
Solvents for which No Adequate Toxicological Data was Found
The following solvents (Table 4) may also be of interest to manufacturers
of excipients, drug substances, or drug products. However, no adequate
toxicological data on which to base a PDE was found. Manufacturers should
supply justification for residual levels of these solvents in pharmaceutical
products.
TABLE 4. Solvents for which no adequate toxicological data was found.
1,1-Diethoxypropane Methylisopropyl
ketone
1,1- Methyltetrahydrofuran
Dimethoxymethane
2,2- Petroleum ether
Dimethoxypropane
Isooctane Trichloroacetic acid
Isopropyl ether Trifluoroacetic acid

5
The information included for Triethylamine reflects that included in the
Revision of PDE Information for Triethylamine which reached Step 4 in
November 2016 and was subsequently incorporated into the core Guideline.
GLOSSARY
Genotoxic Carcinogens:
30
 Carcinogens which produce cancer by affecting genes or chromosomes.
LOEL:
Abbreviation for lowest-observed effect level.
Lowest-Observed Effect Level:
The lowest dose of substance in a study or group of studies that produces
biologically significant increases in frequency or severity of any effects in the
exposed humans or animals.
Modifying Factor:
A factor determined by professional judgment of a toxicologist and applied
to bioassay data to relate that data safely to humans.
Neurotoxicity:
The ability of a substance to cause adverse effects on the nervous system.
NOEL:
Abbreviation for no-observed-effect level.
No-Observed-Effect Level:
The highest dose of substance at which there are no biologically significant
increases in frequency or severity of any effects in the exposed humans or
animals.
PDE:
Abbreviation for permitted daily exposure.
Permitted Daily Exposure:
The maximum acceptable intake per day of residual solvent in
pharmaceutical
products.
Reversible Toxicity:
The occurrence of harmful effects that are caused by a substance and which
disappear after exposure to the substance ends.
Strongly Suspected Human Carcinogen:

31
 A substance for which there is no epidemiological evidence of
carcinogenesis but there are positive genotoxicity data and clear evidence of
carcinogenesis in rodents.
Teratogenicity:
The occurrence of structural malformations in a developing fetus when a
substance is administered during pregnancy.
UNIT-II
ELEMENTAL IMPURITIES
INTRODUCTION
Elemental impurities in drug products may arise from several sources; they
may be residual catalysts that were added intentionally in synthesis or may be
present as impurities (e.g., through interactions with processing equipment or
container/closure systems or by being present in components of the drug
product). Because elemental impurities do not provide any therapeutic benefit to
the patient, their levels in the drug product should be controlled within acceptable
limits.
There are three parts of this guidance:
 The evaluation of the toxicity data for potential elemental impurities;
 The establishment of a permitted daily exposure (PDE) for each element of
toxicological concern;
 And application of a risk-based approach to control elemental impurities in
drug products.
An applicant is not expected to tighten the limits based on process capability,
provided that the elemental impurities in drug products do not exceed the PDEs.
The PDEs established in this guidance are considered to be protective of public
health for all patient populations. In some cases, lower levels of elemental
impurities may be warranted when levels below toxicity thresholds have been
shown to have an impact on other quality attributes of the drug product (e.g.,
element catalyzed degradation of drug substances). In addition, for elements with
high PDEs, other limits may have to be considered from a pharmaceutical quality
perspective and other guidances should be consulted (e.g., ICH Q3A).
This guidance presents a process to assess and control elemental impurities in
the drug product using the principles of risk management as described in ICH
32
Q9. This process provides a platform for developing a risk-based control strategy
to limit elemental impurities in the drug product.
In general, FDA’s guidance documents do not establish legally enforceable
responsibilities.
Instead, guidances describe the Agency’s current thinking on a topic and
should be viewed only as recommendations, unless specific regulatory or
statutory requirements are cited. The use of the word should in Agency guidances
means that something is suggested or recommended, but not required.
SCOPE
The guidance applies to new finished drug products (as defined in ICH
Q6A and Q6B) and new drug products containing existing drug substances. The
drug products containing purified proteins and polypeptides (including proteins
and polypeptides produced from recombinant or nonrecombinant origins), their
derivatives, and products of which they are components (e.g., conjugates) are
within the scope of this guidance, as are drug products containing synthetically
produced polypeptides, polynucleotides, and oligosaccharides.
This guidance does not apply to herbal products, radiopharmaceuticals,
vaccines, cell metabolites, DNA products, allergenic extracts, cells, whole blood,
cellular blood components or blood derivatives including plasma and plasma
derivatives, dialysate solutions not intended for systemic circulation, and
elements that are intentionally included in the drug product for therapeutic
benefit.
This guidance does not apply to products based on genes (gene therapy),
cells (cell therapy), and tissue (tissue engineering). In some regions, these
products are known as advanced therapy medicinal products.
This guidance does not apply to drug products used during clinical research
stages of development. As the commercial process is developed, the principles
contained in this guidance can be useful in evaluating elemental impurities that
may be present in a new drug product. Application of Q3D to existing products is
not expected prior to 36 months after publication of the guideline by ICH.
SAFETY ASSESSMENT OF POTENTIAL ELEMENTAL IMPURITIES
A. Principles of the Safety Assessment of Elemental Impurities for Oral,
Parenteral and Inhalation Routes of Administration (3.1)
33
 The method used for establishing the PDE for each elemental impurity is
discussed in detail in Appendix 1. Elements evaluated in this guidance were
assessed by reviewing the publicly available data contained in scientific journals,
government research reports and studies, international regulatory standards
(applicable to drug products) and guidance, and regulatory authority research and
assessment reports. This process follows the principles described in ICH Q3C
Residual Solvents. The available information was reviewed to establish the oral,
parenteral and inhalation PDEs. For practical purposes, the PDEs to be applied to
the drug product that are presented in Appendix 2, Table A.2.1 have been
rounded to 1 or 2 significant figures.
A summary safety assessment identifying the critical study for setting a
PDE for each element is included in Appendix 3. There are insufficient data to
set PDEs by any route of administration for iridium, osmium, rhodium, and
ruthenium. The PDEs for these elements were established on the basis of their
similarity to palladium.
The factors considered in the safety assessment for establishing the PDE
are listed below in approximate order of relevance:
 The likely oxidation state of the element in the drug product
 Human exposure and safety data when it provided applicable information
 The most relevant animal study
 Route of administration
 The relevant endpoint(s)
Standards for daily intake for some of the elemental impurities discussed in
this guidance exist for food, water, air, and occupational exposure. Where
appropriate, these standards were considered in the safety assessment and
establishment of the PDEs.
The longest duration animal study was generally used to establish the PDE.
When a shorter duration animal study was considered the most relevant, the
rationale is provided in the individual safety assessment.
Inhalation studies using soluble salts (when available) were preferred over
studies using particulates for inhalation safety assessment and derivation of
inhalation PDEs. Depending on available data, inhalation PDEs were based on
either local (respiratory system) or systemic toxicity. For PDEs established for
inhalation (and oral or parenteral routes as applicable), doses were normalized to
a 24-hour, 7-day exposure.
34
 In the absence of data and/or where data are available but not considered
sufficient for a safety assessment for the parenteral and or inhalation route of
administration, modifying factors based on oral bioavailability were used to
derive the PDE from the oral PDE:
 Oral bioavailability <1%: divide by a modifying factor of 100;
 Oral bioavailability ≥ 1% and <50%: divide by a modifying factor of 10;
 Oral bioavailability ≥50% and <90%: divide by a modifying factor of 2;
and
 Oral bioavailability ≥ 90%: divide by a modifying factor of 1.
Where oral bioavailability data or occupational inhalation exposure limits
were not available, a calculated PDE was used based on the oral PDE divided by
a modifying factor of 100 (Ref. 1).
B. Other Routes of Administration (3.2)
PDEs were established for oral, parenteral, and inhalation routes of
administration. When PDEs are necessary for other routes of administration, the
concepts described in this guidance may be used to derive PDEs. An assessment
may either increase or decrease an established PDE. The process of derivation of
the PDE for another route of administration may include the following:
 Consider the oral PDE in Appendix 3 as a starting point in developing a
route-specific PDE. Based on a scientific evaluation, the parenteral and
inhalation PDEs may be a more appropriate starting point.
 Assess if the elemental impurity is expected to have local effects when
administered by the intended route of administration:
 If local effects are expected, assess whether a modification to an established
PDE is necessary.
 Consider the doses/exposures at which these effects can be expected
relative to the adverse effect that was used to set an established PDE.
 If local effects are not expected, no adjustment to an established PDE is
necessary.
 If data are available, evaluate the bioavailability of the element via the
intended route of administration and compare this to the bioavailability of
the element by the route with an established PDE:
 When a difference is observed, a correction factor may be applied to an
established PDE. For example, when no local effects are expected, if the
oral bioavailability of an element is 50 percent and the bioavailability of an
element by the intended route is 10 percent, a correction factor of 5 may be
applied.
35
  If a PDE proposed for the new route is increased relative to an established
PDE, quality attributes may need to be considered.
C. Justification for Elemental Impurity Levels Higher Than an Established
PDE (3.3)
Levels of elemental impurities higher than an established PDE (see Table
A.2.1) may be acceptable in certain cases. These cases could include, but are not
limited to, the following situations:
 Intermittent dosing
 Short term dosing (i.e., 30 days or less)
 Specific indications (e.g., life-threatening, unmet medical needs, rare
diseases)
Examples of justifying an increased level of an elemental impurity using a
subfactor approach of a modifying factor (Refs. 2 and 3) are provided below.
Other approaches may also be used to justify an increased level. Any proposed
level higher than an established PDE should be justified on a case-by-case basis.
Example 1: Element X is present in an oral drug product. From the element X
monograph in Appendix 3, a no-observed-adverse-effect level (NOAEL) of 1.1
milligram (mg)/kilogram (kg)/day was identified. Modifying factors F1-F5 have
been established as 5, 10, 5, 1, and 1, respectively. Using the standard approach
for modifying factors as described in Appendix 1, the PDE is calculated as
follows:
PDE = 1.1 mg/kg/day x 50 kg / 5 x 10 x 5 x 1 x 1 = 220 microgram (μg)/day
Modifying factor F2 (default = 10) can be subdivided into two subfactors, one
for toxicokinetics (TK) and one for toxicodynamics, each with a range from 1 to
3.16. Using the plasma half-life of 5 days, the TK adjustment factor could be
decreased to 1.58 for once weekly administration (~1 half-life), and to 1 for
administration once a month (~5 half-lives). Using the subfactor approach for F2,
the proposed level for element X administered once weekly can be calculated as
follows:
Proposed level = 1.1 mg/kg/d x 50 kg / 5 x (1.6 x 3.16) x 5 x 1 x 1 = 440
μg/day
For practical purposes, this value is rounded to 400 μg/day.
Example 2: The TK adjustment factor approach may also be appropriate for
elemental impurities that were not developed using the modifying factor
36
approach. For element Z, a minimal risk level (MRL) of 0.02 mg/kg/day was
used to derive the oral PDE. From literature sources, the plasma half-life was
reported to be 4 days. This element is an impurity in an oral drug product
administered once every 3 weeks (~ 5 half-lives). Using first-order kinetics, the
established PDE of 1000 μg/day is modified as follows:
Proposed level = 0.02 mg/kg/day x 50 kg / 1/3.16 = 3.16 mg/day
For practical purposes, this value is rounded to 3000 μg/day.
D. Parenteral Products (3.4)
For parenteral drug products with maximum daily volumes up to 2 liters,
the maximum daily volume should be used to calculate permissible
concentrations from PDEs. For products whose daily volumes, as specified by
labeling and/or established by clinical practice, may exceed 2 liters (e.g., saline,
dextrose, total parenteral nutrition, solutions for irrigation), a 2-liter volume may
be used to calculate permissible concentrations from PDEs. (Ref. 4)
ELEMENT CLASSIFICATION (4)
The elements included in this guidance have been placed into three classes
based on their toxicity (PDE) and likelihood of occurrence in the drug product.
The likelihood of occurrence is derived from several factors including:
probability of use in pharmaceutical processes, probability of being a co-isolated
impurity with other elemental impurities in materials used in pharmaceutical
processes, and the observed natural abundance and environmental distribution of
the element.
For the purposes of this guidance, an element with low natural abundance
refers to an element with a reported natural abundance of < 1 atom/106 atoms of
silicon (Ref. 5). The classification scheme is intended to focus the risk
assessment on those elements that are the most toxic but also have a reasonable
probability of inclusion in the drug product (see Table V.1 (5.1)). The elemental
impurity classes are:
Class 1: The elements arsenic (As), cadmium (Cd), mercury (Hg), and lead (Pb)
are human toxicants that have limited or no use in the manufacture of
pharmaceuticals. Their presence in drug products typically comes from
commonly used materials (e.g., mined excipients). Because of their unique
nature, these four elements should be evaluated during the risk assessment,
across all potential sources of elemental impurities and routes of administration.
37
The outcome of the risk assessment will determine those components that may
require additional controls, which may in some cases include testing for Class 1
elements. It is not expected that all components will require testing for Class 1
elemental impurities; testing should only be applied when the risk assessment
identifies it as the appropriate control to ensure that the PDE will be met.
Class 2: Elements in this class are generally considered as route-dependent
human toxicants.
Class 2 elements are further divided in sub-classes 2A and 2B based on
their relative likelihood of occurrence in the drug product.
Class 2A elements have relatively high probability of occurrence in the drug
product, thus should be evaluated in the risk assessment across all potential
sources of elemental impurities and routes of administration (as indicated). The
class 2A elements are: cobalt (Co), nickel (Ni), and vanadium (V).
Class 2B elements have a reduced probability of occurrence in the drug product
related to their low abundance and low potential to be co-isolated with other
materials. As a result, they can be excluded from the risk assessment unless they
are intentionally added during the manufacture of drug substances, excipients or
other components of the drug product. The elemental impurities in class 2B
include: silver (Ag), gold (Au), iridium (Ir), osmium (Os), palladium (Pd),
platinum (Pt), rhodium (Rh), ruthenium (Ru), selenium (Se), and thallium (Tl).
Class 3: The elements in this class have relatively low toxicities by the oral route
of administration (high PDEs, generally > 500 μg/day) but could warrant
consideration in the risk assessment for inhalation and parenteral routes. For oral
routes of administration, unless these elements are intentionally added, they do
not need to be considered during the risk assessment. For parenteral and
inhalation products, the potential for inclusion of these elemental impurities
should be evaluated during the risk assessment, unless the route specific PDE is
above 500 μg/day. The elements in this class include: barium (Ba), chromium
(Cr), copper (Cu), lithium (Li), molybdenum (Mo), antimony (Sb), and tin (Sn).
Other elements: Some elemental impurities for which PDEs have not been
established due to their low inherent toxicity and/or differences in regional
regulations are not addressed in this guidance. If these elemental impurities are
present or included in the drug product they are addressed by other guidances
and/or regional regulations and practices that may be applicable for particular
elements (e.g., aluminium for compromised renal function; manganese and zinc
38
for patients with compromised hepatic function), or quality considerations (e.g.,
presence of tungsten impurities in therapeutic proteins) for the final drug product.
Some of the elements considered include: aluminium (Al), boron (B), calcium
(Ca), iron (Fe), potassium (K), magnesium (Mg), manganese (Mn), sodium (Na),
tungsten (W), and zinc (Zn).
RISK ASSESSMENT AND CONTROL OF ELEMENTAL IMPURITIES
In developing controls for elemental impurities in drug products, the
principles of quality risk management, described in ICH Q9, should be
considered. The risk assessment should be based on scientific knowledge and
principles. It should link to safety considerations for patients with an
understanding of the product and its manufacturing process (ICH Q8 and Q11).
In the case of elemental impurities, the product risk assessment would therefore
be focused on assessing the levels of elemental impurities in a drug product in
relation to the PDEs presented in this guidance.
Information for this risk assessment includes but is not limited to: data
generated by the applicant, information supplied by drug substance and/or
excipient manufacturers, and/or data available in published literature.
The applicant should document the risk assessment and control approaches
in an appropriate manner. The level of effort and formality of the risk assessment
should be proportional to the level of risk. It is neither always appropriate nor
always necessary to use a formal risk management process (using recognized
tools and/or formal procedures, e.g., standard operating procedures). The use of
informal risk management processes (using empirical tools and/or internal
procedures) may also be considered acceptable. Tools to assist in the risk
assessment are described in ICH Q8 and Q9 and will not be presented in this
guidance.
A. General Principles
For the purposes of this guidance, the risk assessment process can be
described in three steps:
 Identify known and potential sources of elemental impurities that may find
their way into the drug product.
 Evaluate the presence of a particular elemental impurity in the drug product
by determining the observed or predicted level of the impurity and
comparing with the established PDE.

39
  Summarize and document the risk assessment. Identify if controls built into
the process are sufficient, or identify additional controls to be considered to
limit elemental impurities in the drug product.
In many cases, the steps are considered simultaneously. The outcome of the
risk assessment may be the result of iterations to develop a final approach to
ensure the potential elemental impurities do not exceed the PDE.
B. Potential Sources of Elemental Impurities
In considering the production of a drug product, there are broad categories
of potential sources of elemental impurities.
 Residual impurities resulting from elements intentionally added (e.g.,
catalysts) in the formation of the drug substance, excipients, or other drug
product components. The risk assessment of the drug substance should
address the potential for inclusion of elemental impurities in the drug
product.
 Elemental impurities that are not intentionally added and are potentially
present in the drug substance, water, or excipients used in the preparation of
the drug product.
 Elemental impurities that are potentially introduced into the drug substance
and/or drug product from manufacturing equipment.
 Elemental impurities that have the potential to be leached into the drug
substance and drug product from container closure systems.
The following diagram shows an example of typical materials, equipment, and
components used in the production of a drug product. Each of these sources may
contribute elemental impurities to the drug product, through any individual or
any combination of the potential sources listed above.
During the risk assessment, the potential contributions from each of these
sources should be considered to determine the overall contribution of elemental

40
impurities to the drug product.

* The risk of inclusion of elemental impurities can be reduced through

process understanding, equipment selection, equipment qualification and Good
Manufacturing Practice (GMP) processes.
** The risk of inclusion of elemental impurities from water can be reduced by
complying with compendial (e.g.,
European Pharmacopoeia, Japanese Pharmacopoeia, US Pharmacopeial
Convention) water quality requirements, if purified water or water for injection is
used in the manufacturing process(es).
C. Identification of Potential Elemental Impurities
Potential elemental impurities derived from intentionally added catalysts
and inorganic reagents: If any element listed in Table V.1 (5.1) is intentionally
added, it should be considered in the risk assessment. For this category, the
identity of the potential impurities is known and techniques for controlling the
elemental impurities are easily characterized and defined.
Potential elemental impurities that may be present in drug substances
and/or excipients: While not intentionally added, some elemental impurities may
be present in some drug substances and/or excipients. The possibility for
inclusion of these elements in the drug product should be reflected in the risk
assessment.
For the oral route of administration, the risk assessment should evaluate the
possibility for inclusion of Class 1 and Class 2A elemental impurities in the drug
product. For parenteral and inhalation routes of administration, the risk
assessment should evaluate the possibility for inclusion of the Class 1, Class 2A,
and Class 3 elemental impurities as shown in Table V.1 (5.1).
Potential elemental impurities derived from manufacturing equipment: The
contribution of elemental impurities from this source may be limited, and the
subset of elemental impurities that should be considered in the risk assessment
will depend on the manufacturing equipment used in the production of the drug
product. Application of process knowledge, selection of equipment, equipment
qualification, and GMP controls ensure a low contribution from manufacturing
equipment. The specific elemental impurities of concern should be assessed

41
based on knowledge of the composition of the components of the manufacturing
equipment that come in contact with components of the drug product. The risk
assessment of this source of elemental impurities is one
that can potentially be utilized for many drug products using similar process
trains and processes.
In general, the processes used to prepare a given drug substance are
considerably more aggressive than processes used in preparing the drug product
when assessed relative to the potential to leach or remove elemental impurities
from manufacturing equipment. Contributions of elemental impurities from drug
product processing equipment would be expected to be lower than contributions
observed for the drug substance. However, when this is not the case based on
process knowledge or understanding, the applicant should consider the potential
for incorporation of elemental impurities from the drug product manufacturing
equipment in the risk assessment (e.g., hot melt extrusion).
Elemental impurities leached from container closure systems: The
identification of potential elemental impurities that may be introduced from
container closure systems should be based on a scientific understanding of likely
interactions between a particular drug product type and its packaging. When a
review of the materials of construction demonstrates that the container closure
system does not contain elemental impurities, no additional risk assessment
needs to be performed. It is recognized that the probability of elemental leaching
into solid dosage forms is minimal and does not require further consideration in
the risk assessment. For liquid and semisolid dosage forms, there is a higher
probability that elemental impurities could leach from the container closure
system during the shelf-life of the product. Studies to understand potential
leachables from the container closure system (after washing, sterilization,
irradiation, etc.) should be performed. This source of elemental impurities will
typically be addressed during evaluation of the container closure system for the
drug product.
Factors that should be considered (for liquid and semisolid dosage forms)
include but are not limited to:
 Hydrophilicity/hydrophobicity
 Ionic content
 pH
 Temperature (cold chain vs room temperature and processing conditions)
42
  Contact surface area
 Container/component composition
 Terminal sterilization
 Packaging process
 Component sterilization
 Duration of storage
D. Recommendations for Elements to be Considered in the Risk Assessment
The following table provides recommendations for inclusion of elemental
impurities in the risk assessment. This table can be applied to all sources of

elemental impurities in the drug product.

E. Evaluation
As the potential elemental impurity identification process is concluded,
there are two possible outcomes:

43
 1. The risk assessment process does not identify any potential elemental
impurities. The conclusion of the risk assessment and supporting information and
data should be documented.
2. The risk assessment process identifies one or more potential elemental
impurities. For any elemental impurities identified in the process, the risk
assessment should consider if there are multiple sources of the identified
elemental impurity or impurities and document the conclusion of the assessment
and supporting information.
The applicant’s risk assessment can be facilitated with information about
the potential elemental impurities provided by suppliers of drug substances,
excipients, container closure systems, and manufacturing equipment. The data
that support this risk assessment can come from a number of sources that include,
but are not limited to:
 Prior knowledge
 Published literature
 Data generated from similar processes;
 Supplier information or data
 Testing of the components of the drug product
 Testing of the drug product
During the risk assessment, there are a number of factors that can influence
the level of the potential impurity in the drug product and should also have been
considered in the risk assessment.
These include but are not limited to:
 Efficiency of removal of elemental impurities during further processing
 Natural abundance of elements (especially important for the categories of
elements that are not intentionally added)
 Prior knowledge of elemental impurity concentration ranges from specific
sources
 The composition of the drug product
F. Summary of Risk Assessment Process
The risk assessment is summarized by reviewing relevant product or
component specific data combined with information and knowledge gained
across products or processes to identify the significant probable elemental
impurities that may be observed in the drug product.

44
 The summary should consider the significance of the observed or predicted
level of the elemental impurity relative to the PDE of the elemental impurity. As
a measure of the significance of the observed elemental impurity level, a control
threshold is defined as a level that is 30 percent of the established PDE in the
drug product. The control threshold may be used to determine if additional
controls are warranted.
If the total elemental impurity level from all sources in the drug product is
expected to be consistently less than 30 percent of the PDE, then additional
controls are not required, provided the applicant has appropriately assessed the
data and demonstrated adequate controls on elemental impurities.
If the risk assessment fails to demonstrate that an elemental impurity level
is consistently less than the control threshold, controls should be established to
ensure that the elemental impurity level does not exceed the PDE in the drug
product. (See section VI (6).)
The variability of the level of an elemental impurity should be factored into
the application of the control threshold to drug products. Sources of variability
may include:
 Variability of the analytical method
 Variability of the elemental impurity level in the specific sources
 Variability of the elemental impurity level in the drug product
At the time of submission, in the absence of other justification, the level and
variability of an elemental impurity can be established by providing the data
from three (3) representative production scale lots or six (6) representative pilot
scale lots of the component or components or drug product. For some
components that have inherent variability (e.g., mined excipients), additional data
may be needed to apply the control threshold.
There are many acceptable approaches to summarizing and documenting the
risk assessment, including: tables, written summaries of considerations and
conclusions of the assessment. The summary should identify the elemental
impurities, their sources, and the controls and acceptance criteria as needed.
G. Special Considerations for Biotechnologically Derived Products
For biotechnologically derived products, the risks of elemental impurities
being present at levels that raise safety concerns at the drug substance stage are
considered low. This is largely because:

45
 1. Elements are not typically used as catalysts or reagents in the
manufacturing of biotech products.
2. Elements are added at trace levels in media feeds during cell culture
processes, without accumulation and with significant dilution/removal during
further processing.
3. Typical purification schemes used in biotech manufacturing such as
extraction, chromatography steps and dialysis or ultrafiltration-diafiltration
(UF/DF) have the capacity to clear elements introduced in cell
culture/fermentation steps or from contact with manufacturing equipment to
negligible levels.
As such, specific controls on elemental impurities up to the biotech drug
substance are generally not needed. In cases where the biotechnologically
derived drug substance contains synthetic structures (such as antibody-drug
conjugates), appropriate controls on the small molecule component for elemental
impurities should be evaluated.
However, potential elemental impurity sources included in drug product
manufacturing (e.g., excipients) and other environmental sources should be
considered for biotechnologically derived drug products. The contribution of
these sources to the finished product should be assessed because they are
typically introduced in the drug product manufacture at a step in the process
where subsequent elemental impurity removal is not generally performed. Risk
factors that should be considered in this assessment should include the type of
excipients used, the processing conditions and their susceptibility to
contamination by environmental factors (e.g., controlled areas for sterile
manufacturing and use of purified water), and overall dosing frequency.
CONTROL OF ELEMENTAL IMPURITIES
Control of elemental impurities is one part of the overall control strategy
for a drug product that assures that elemental impurities do not exceed the PDEs.
When the level of an elemental impurity may exceed the control threshold,
additional measures should be implemented to assure that the level does not
exceed the PDE. Approaches that an applicant can pursue include but are not
limited to:

46
  Modification of the steps in the manufacturing process that result in the
reduction of elemental impurities below the control threshold through
specific or non-specific purification steps
 Implementation of in-process or upstream controls, designed to limit the
concentration of the elemental impurity below the control threshold in the
drug product
 Establishment of specification limits for excipients or materials (e.g.,
synthetic intermediates)
 Establishment of specification limits for the drug substance
 Establishment of specification limits for the drug product
 Selection of appropriate container closure systems
Periodic testing may be applied to elemental impurities according to the
principles described in ICH Q6A.
The information on the control of elemental impurities that is provided in a
regulatory submission includes, but is not limited to, a summary of the risk
assessment, appropriate data as necessary, and a description of the controls
established to limit elemental impurities.
CONVERTING BETWEEN PDES AND CONCENTRATION LIMITES
The PDEs, reported in micrograms per day (μg/day), provided in this
document give the maximum permitted quantity of each element that may be
contained in the maximum daily intake of a drug product. Because the PDE
reflects only total exposure from the drug product, it is useful to convert the PDE
into concentrations as a tool in evaluating elemental impurities in drug products
or their components. The options listed in this section describe some acceptable
approaches to establishing concentrations of elemental impurities in drug
products or components that would assure that the drug product does not exceed
the PDEs. The applicant may select any of these options as long as the resulting
permitted concentrations assure that the drug product does not exceed the PDEs.
In the choice of a specific option, the applicant must have knowledge of, or make
assumptions about, the daily intake of the drug product. The permitted
concentration limits may be used:
 As a tool in the risk assessment to compare the observed or predicted levels
to the PDE;
 In discussions with suppliers to help establish upstream controls that would
assure that the product does not exceed the PDE;
 To establish concentration targets when developing in-process controls on
elemental impurities;
47
  To convey information regarding the controls on elemental impurities in
regulatory submissions.
There are multiple sources of elemental impurities in drug products. When
applying any of the options described below, elemental impurities from container
closure systems and manufacturing equipment should be taken into account
before calculating the maximum permitted concentration in the remaining
components (excipients and drug substance). If it is determined during the risk
assessment that the container closure systems and manufacturing equipment do
not contribute to the elemental impurity level in the drug product, they do not
need to be considered. Where contributions from container closure systems and
manufacturing equipment exist, these contributions may be accounted for by
subtracting the estimated daily intake from these sources from the PDE before
calculation of the allowed concentration in the excipients and drug substance.
Option 1: Common permitted concentration limits of elements across drug
product components for drug products with daily intakes of not more than 10
grams: This option is not intended to imply that all elements are present at the
same concentration, but rather provides a simplified approach to the calculations.
The option assumes the daily intake (amount) of the drug product is 10
grams or less, and that elemental impurities identified in the risk assessment (the
target elements) are present in all components of the drug product. Using
Equation 1 below, and a daily intake of 10 grams of drug product, this option
calculates a common permissible target elemental concentration for each
component in the drug. This approach, for each target element, allows
determination of a fixed common maximum concentration in micrograms per
gram in each component. The permitted concentrations are provided in Appendix
2, Table A.2.2.

If all the components in a drug product do not exceed the Option 1

concentrations for all target elements identified in the risk assessment, then all
these components may be used in any proportion in the drug product. An
example using this option is shown in Appendix 4, Table A.4.2. If the permitted
48
concentrations in Appendix 2, Table A.2.2 are not applied, Options 2a, 2b, or 3
should be followed.
Option 2a: Common permitted concentration limits across drug product
components for a drug product with a specified daily intake: This option is
similar to Option 1, except that the drug daily intake is not assumed to be 10
grams. The common permitted concentration of each element is determined using
Equation 1 and the actual maximum daily intake.
This approach, for each target element, allows determination of a fixed
common maximum concentration in micrograms per gram in each component
based on the actual daily intake provided. An example using this option is
provided in Appendix 4, Table A.4.3.
If all components in a drug product do not exceed the Option 2a
concentrations for all target elements identified in the risk assessment, then all
these components may be used in any proportion in the drug product.
Option 2b: Permitted concentration limits of elements in individual components
of a product with a specified daily intake: This option should be supported with
additional information that the applicant may assemble regarding the potential
for specific elemental impurities to be present in specific drug product
components. The applicant may set permitted concentrations based on the
distribution of elements in the components (e.g., higher concentrations in
components with the presence of an element in question). For each element
identified as potentially present in the components of the drug product, the
maximum expected mass of the elemental impurity in the final drug product can
be calculated by multiplying the mass of each component material times the
permitted concentration established by the applicant in each material and
summing over all components in the drug product, as described in Equation 2.
The total mass of the elemental impurity in the drug product should comply with
the PDEs given in Appendix 2, Table A.2.1 unless justified according to other
relevant sections of this guidance. If the risk assessment has determined that a
specific element is not a potential impurity in a specific component, there is no
need to establish a quantitative result for that element in that component. This
approach allows that the maximum permitted concentration of an element in
certain components of the drug product may be higher than the Option 1 or
Option 2a limit, but this should then be compensated by lower allowable
concentrations in the other components of the drug product. Equation 2 may be

49
used to demonstrate that component-specific limits for each element in each
component of a drug product assure that the PDE will be met.

An example using this option is provided in Appendix 4, Tables A.4.4 –

A.4.5.
Option 3: Finished Product Analysis:
The concentration of each element may be measured in the final drug
product. Equation 1 may be used with the maximum total daily dose of the drug
product to calculate a maximum permitted concentration of the elemental
impurity. An example using this option is provided in Appendix 4, Table A.4.6.
SPECIATION AND OTHER CONSIDERATIONS
Speciation is defined as the distribution of elements among chemical
species including isotopic composition, electronic or oxidation state, and/or
complex or molecular structure. When the toxicities of different species of the
same element are known, the PDE has been established using the toxicity
information on the species expected to be in the drug product.
When elemental impurity measurements are used in the risk assessment,
total elemental impurity levels in drug products may be used to assess
compliance with the PDEs. The applicant is not expected to provide speciation
information; however, such information could be used to justify lower or higher
levels when the identified species is more or less toxic, respectively, than the
species used in the monographs in Appendix 3.
When total elemental impurity levels in components are used in the risk
assessment, the applicant is not expected to provide information on release of an
elemental impurity from the component in which it is found. However, such
information could be used to justify levels higher than those based on the total
elemental impurity content of the drug product.

50
ANALYTICAL PROCEDURES
The determination of elemental impurities should be conducted using
appropriate procedures suitable for their intended purposes. Unless otherwise
justified, the test should be specific for each elemental impurity identified for
control during the risk assessment. Pharmacopoeial procedures or suitable
alternative procedures for determining levels of elemental impurities should be
used.
LIFECYCLE MANAGEMENT
The quality systems and management responsibilities described in ICH Q10
are intended to encourage the use of science-based and risk-based approaches at
each lifecycle stage, thereby promoting continual improvement across the entire
product lifecycle. Product and process knowledge should be managed from
development through the commercial life of the product up to and including
product discontinuation.
Knowledge gained from development combined with commercial
manufacturing experience and data can be used to further improve process
understanding and process performance. Such improvements can enhance
controls on elemental impurities. It is recognized that the elemental impurity data
available for some components is somewhat limited at the date of publication of
this guidance, which may direct the applicant to a specific set of controls.
Additional data, if developed, may lead to modifications of the controls.
If changes to the drug product or components have the potential to change
the elemental impurity content of the drug product, the risk assessment, including
established controls for elemental impurities, should be re-evaluated. Such
changes could include, but are not limited to: changes in synthetic routes,
excipient suppliers, raw materials, processes, equipment, container closure
systems, or facilities. All changes are subject to internal change management
process (ICH Q10) and, if needed, appropriate regional regulatory requirements.
GLOSSARY
ACGIH: American Conference of Governmental Industrial Hygienists.
ATSDR: Agency for Toxic Substances and Disease Registry.
CEC: Commission of the European Community.
CFR: Code of Federal Regulations (USA).
51
Change management: A systematic approach to proposing, evaluating,
approving, implementing and reviewing changes (ICH Q10).
CICAD: Concise International Chemical Assessment Documents (WHO).
Container closure system: The sum of packaging components that together
contain and protect the dosage form. This includes primary packaging
components and secondary packaging components, if the latter are intended to
provide additional protection to the drug product. A packaging system is
equivalent to a container closure system (ICH Q1A).
Control strategy: A planned set of controls, derived from current product and
process understanding, that assures process performance and product quality. The
controls can include parameters and attributes related to drug substance and drug
product materials and components, facility and equipment operating conditions,
in-process controls, finished product specifications, and the associated methods
and frequency of monitoring and control (ICH Q10).
Control threshold: A limit that is applied during the assessment of elemental
impurities to determine if additional control elements may be required to ensure
that the PDE is not exceeded in the drug product. The limit is defined as 30
percent of the PDE of the specific elemental impurity under consideration.
Daily dose: The total mass of drug product that is consumed by a patient on a
daily basis.
EFSA: European Food Safety Agency.
EHC: Environmental Health Criteria (IPCS, WHO).
EU SCOEL: European Scientific Committee on Occupational Exposure Limits.
EU SEG: European Union Scientific Expert Group.
Herbal products: Medicinal products containing, exclusively, plant material
and/or vegetable drug preparations as active ingredients. In some traditions,
materials of inorganic or animal origin can also be present.
IARC: International Agency for Research on Cancer.
Inhalation unit risk: The upper-bound excess lifetime cancer risk estimated to
result from continuous exposure to an agent at a concentration of 1 μg/liter (L) in
water, or 1 μg/m3 in air. The interpretation of inhalation unit risk would be as
follows: if unit risk = 2 x 10-6 per μg/L, 2 excess cancer cases (upper bound
52
estimate) are expected to develop per 1,000,000 people if exposed daily for a
lifetime to 1 μg of the chemical in 1 liter of drinking water (US EPA).
IPCS: International Programme for Chemical Safety.
IUPAC: International Union of Pure and Applied Chemistry.
IRIS: Integrated Risk Identification System, United States Environmental
Protection Agency.
LOAEL: Lowest-observed-adverse-effect level: Lowest concentration or amount
of a substance (dose), found by experiment or observation, that causes an adverse
effect on morphology, functional capacity, growth, development, or life span of a
target organism distinguishable from normal (control) organisms of the same
species and strain under defined conditions of exposure (IUPAC).
LoQ: Limit of quantitation: The quantitation limit of an individual analytical
procedure is the lowest amount of analyte in a sample which can be
quantitatively determined with suitable precision and accuracy. The quantitation
limit is a parameter of quantitative assays for low levels of compounds in sample
matrices, and is used particularly for the determination of impurities and/or
degradation products (ICH Q2).
LOEL: Lowest-observed-effect level: The lowest dose of substance in a study or
group of studies that produces biologically significant increases in frequency or
severity of any effects in the exposed humans or animals.
Modifying factor: An individual factor determined by professional judgment of
a toxicologist and applied to bioassay data to relate that data to human safety
(ICH Q3C) (see related term Safety factor).
MRL: Minimal risk level: An estimate of the daily human exposure to a
hazardous substance that is likely to be without appreciable risk (ATSDR).
NAS: National Academy of Science (USA).
NOAEL: No-observed-adverse-effect level: Greatest concentration or amount of
a substance, found by experiment or observation, that causes no detectable
adverse alteration of morphology, functional capacity, growth, development, or
life span of the target organism under defined conditions of exposure.
NOEL: No-observed-effect level: The highest dose of substance at which there
are no biologically significant increases in frequency or severity of any effects in
the exposed humans or animals.
53
NTP: National Toxicology Program (USA).
OEHHA: Office of Environmental Health Hazard Assessment (California,
USA).
OELV: Occupational Exposure Limit Value.
OSHA: Occupational Safety and Health Administration (USA).
PEL: Permissible exposure limit.
PDE: Permitted daily exposure: The maximum acceptable intake of elemental
impurity in pharmaceutical products per day.
Product lifecycle: All phases in the life of the product from the initial
development through marketing until the product’s discontinuation (ICH Q9).
Quality: The degree to which a set of inherent properties of a product, system, or
process fulfils requirements (see ICH Q6A definition specifically for quality of
drug substance and drug products) (ICH Q9).
Quality risk management: A systematic process for the assessment, control,
communication, and review of risks to the quality of the drug product across the
product lifecycle (ICH Q9).
Quality system: The sum of all aspects of a system that implements quality
policy and ensures that quality objectives are met (ICH Q10).
Risk: The combination of the probability of occurrence of harm and the severity
of that harm (ISO/IEC Guide 51, ICH Q9).
Risk acceptance: The decision to accept risk (ISO Guide 73).
Risk analysis: The estimation of the risk associated with the identified hazards
(ICH Q9).
Risk assessment: A systematic process of organizing information to support a
risk decision to be made within a risk management process. It consists of the
identification of hazards and the analysis and evaluation of risks associated with
exposure to those hazards (ICH Q9).
Risk control: Actions implementing risk management decisions (ISO Guide 73).
Risk identification: The systematic use of information to identify potential
sources of harm (hazards) referring to the risk question or problem description
(ICH Q9).
54
Risk management: The systematic application of quality management policies,
procedures, and
practices to the tasks of assessing, controlling, communicating, and reviewing
risk (ICH Q9).
Safety: Practical certainty that adverse effects will not result from exposure to an
agent under defined circumstances (Ref. 2).
Safety assessment: An approach that focuses on the scientific understanding and
measurement of chemical hazards as well as chemical exposures, and ultimately
the risks associated with them. This term is often (and in this guidance) used
synonymously with risk assessment (Ref. 2).
Safety factor: A composite (reductive) factor applied by the risk assessment
experts to the NOAEL or other reference point, such as the benchmark dose or
benchmark dose lower confidence limit, to derive a reference dose that is
considered safe or without appreciable risk, such as an acceptable daily intake or
tolerable daily intake (the NOAEL or other reference point is divided by the
safety factor to calculate the reference dose). The value of the safety factor
depends on the nature of the toxic effect, the size and type of population to be
protected, and the quality of the toxicological information available. See related
terms: Assessment factor, Uncertainty factor. (Ref. 2)
Severity: A measure of the possible consequences of a hazard (ICH Q9).
TLV: Threshold limit value: The concentration in air to which it is believed that
most workers can be exposed daily without an adverse effect (i.e., effectively, the
threshold between safe and dangerous concentrations). The values were
established (and are revised annually) by the ACGIH and are time-weighted
concentrations (TWA) for a 7- or 8-hour workday and 40-hour workweek, and
thus related to chronic effects (IUPAC).
TWA: Time Weighted Average: As defined by ACGIH, time-weighted average
concentration for a conventional 8-hour workday and a 40-hour workweek
(IUPAC).
URF: Unit Risk Factor.
US DoL: United States Department of Labor.
US EPA: United States Environmental Protection Agency.
WHO: World Health Organization.
55
ELEMENTAL ANALYSIS
Elemental analysis is a process where a sample of some material (e.g.,
soil, waste or drinking water, bodily fluids, minerals, chemical compounds) is
analyzed for its elemental and sometimes isotopic composition. Elemental
analysis can be qualitative (determining what elements are present), and it can be
quantitative (determining how much of each are present). Elemental analysis falls
within the ambit of analytical chemistry, the set of instruments involved in
deciphering the chemical nature of our world.

For organic chemists, elemental analysis or "EA" almost always refers to

CHNX analysis—the determination of the mass fractions of carbon, hydrogen,
nitrogen, and heteroatoms (X) (halogens, sulfur) of a sample. This information
was very important to help determine the structure of an unknown compound, as
well as to help ascertain the structure and purity of a synthesized compound. In
present day organic chemistry spectroscopic technics (like NMR, both 1H and
13
C), mass spectrometry and chromatographic procedures have replaced EA as
the primary technique for structural determination, although it still gives very
useful complementary information. It is also the fastest and most inexpensive
method to determine sample purity.

Antoine Lavoisier is regarded as the inventor of elemental analysis as a

quantitate, experimental tool to assess the chemical composition of a compound.
At the time elemental analysis was based on gravimetric determination of
specific adsorbant materials before and after selective adsorption of the
combustion gases. Today fully automated systems based on thermal conductivity
or infrared spectroscopy detection of the combustion gases, or other
spectroscopic methods are used.

Elemental analysis on carbon, hydrogen and nitrogen is the most essential

- and in many cases the only - investigation performed to characterize and/or
prove the elemental composition of an organic sample. Numerous compounds
include no additional elements besides C, H and N except oxygen, which is
seldom determined seperately (although it can be done!).

Determination of C/H/N is done using a "2400 CHN Elemental Analyzer"

by Perkin Elmer. This apparatus was introduced in 1989. Prior C/H/N-analysis
was performed on the precursor model "240 CHN Elemental Analyzer"
developed by the same supplier. This instrument is now in use for the oxygen
determination.

56
CHNS Elemental Analysers

CHNS elemental analysers provide a means for the rapid determination of

carbon, hydrogen, nitrogen and sulphur in organic matrices and other types of
materials. They are capable of handling a wide variety of sample types, including
solids, liquids, volatile and viscous samples, in the fields of pharmaceuticals,
polymers, chemicals, environment, food and energy.
The analysers are often constructed in modular form such that they can be
set up in a number of different configurations to determine, for example, CHN,
CHNS, CNS or N depending on the application. This adaptability allows not only
flexibility of operation but also the use of a wide range of sample weights from a
fraction of a milligram to several grams (macro-systems.)
In its simplest form, simultaneous CHNS analysis requires high
temperature combustion in an oxygen-rich environment and is based on the
classical Pregl-Dumas method. This combustion can be carried out under both
static conditions i.e. introduction of a set volume of oxygen or dynamic
conditions i.e. a constant flow of oxygen for a set period of time. Often, catalysts
are also added to the combustion tube in order to aid conversion.
Basic principles
In the combustion process (furnace at ca. 1000oC), carbon is converted to
carbon dioxide; hydrogen to water; nitrogen to nitrogen gas/ oxides of nitrogen
and sulphur to sulphur dioxide. If other elements such as chlorine are present,

57
they will also be converted to combustion products, such as hydrogen chloride. A
variety of absorbents are used to remove these additional combustion products as
well as some of the principal elements, sulphur for example, if no determination
of these additional elements is required.
The combustion products are swept out of the combustion chamber by inert
carrier gas such as helium and passed over heated (about 600 o C) high purity
copper. This copper can be situated at the base of the combustion chamber or in a
separate furnace. The function of this copper is to remove any oxygen not
consumed in the initial combustion and to convert any oxides of nitrogen to
nitrogen gas. The gases are then passed through the absorbent traps in order to
leave only carbon dioxide, water, nitrogen and sulphur dioxide.
Detection of the gases can be carried out in a variety of ways including (i) a
GC separation followed by quantification using thermal conductivity detection
(ii) a partial separation by GC (‘frontal chromatography’) followed by thermal
conductivity detection (CHN but not S) (iii) a series of separate infra-red and
thermal conductivity cells for detection of individual compounds. Quantification
of the elements requires calibration for each element by using high purity ‘micro-
analytical standard’ compounds such as acetanilide and benzoic acid.
CHNS instrumentation
Combustion elemental analysers are manufactured in a variety of
configurations to suit specific applications, and the choice will depend on the
elements of interest, the sample type and size, and the concentration of the
analyte.
All instruments require two gas supplies: (i) an inert carrier gas (helium
recommended); and (ii) high purity oxygen (minimum 99.9995%). The strict
specification for oxygen is associated with the need to reduce the nitrogen
‘blank’ contribution to an inconsequential level. Additionally, GC-type gas filters
are also usually fitted to prevent trace organic species and water entering the
combustion system.
The choice of sample introduction systems will depend on the application
and the sample type. For solids or viscous liquids, samples are weighed out into
tin capsules; for liquids, samples can be sealed in individual aluminium vials or
introduced via a liquid auto-sampler. Both capsules and vials are pre-cleaned and
dried to avoid trace contamination from oils and water acquired during their
manufacture. Instruments are marketed with either simple ‘one shot’ introduction
58
interfaces or a carousel type autosampler. In some instances a microbalance is
directly interfaced with the analyser to allow the automatic recording of the
weight of each test portion.
The combustion section of the analyser is designed to achieve both
complete combustion of the sample and conversion of oxides of nitrogen to
nitrogen gas (N2). Although different approaches have been chosen by different
manufacturers, the use of high purity copper is universal for the reduction stage.
In some instruments, the combustion and reduction stages are housed in separate
furnaces. In others, the reactions are combined in a single two-tier furnace.
Catalysts are usually added to the combustion section to aid complete
combustion and absorbents to remove potential contaminants. Both the
catalysts/absorbents and copper metal are packed into readily exchangeable tubes
made of ceramic material or high quality silica.
Instruments are classified as either ‘static’ or ‘dynamic’ in terms of their
combustion characteristics. In the ‘static’ type, a pre-set volume of oxygen is
added to the combustion tube before the sample is introduced. In the ‘dynamic’
type, the oxygen is added to the tube at the same time as the sample introduction
and continues to flow for a set time. In the majority of applications, either
method is applicable. For slow burning materials such as coals and cokes, where
multiple additions of oxygen are required for complete combustion, the ‘static’
system is preferred.
The detection system within the analyser can take several forms depending
on the combustion mode and sample size. With small test portions, the
combustion gases can be separated on a GC column and quantified using a
thermal conductivity detector. A schematic diagram of such a system is shown
overleaf. If larger test portions are required, an instrument employing ‘frontal’
chromatography can be chosen. The latter approach employs a GC column with
thermal conductivity detection but provides a step-wise profile for integration.
Yet other detection approaches require no separation step but use separate infra-
red and thermal conductivity cells to respond to individual elements.
Control of the instrument is established through a computer module, which
is used to set up the program of work, report instrument diagnostics, and manage
the calibration procedures.
Further considerations

59
 The choice of instrument depends on a number of factors associated with
the sample type. In instances where obtaining a homogeneous sample can prove
difficult, food analysis for example, a greater weight of test material is required
to provide a representative test portion. The larger the test portions and the higher
the content of organic matter, the more oxygen will be needed to carry out the
combustion successfully. This in turn means that larger capacity reduction tubes
are needed to remove the excess oxygen and provide capacity for a reasonable
number of combustions before replacement. For such applications, a macro-
analyser would be required for gram-sized samples. For less heterogeneous
samples, a micro-type analyser designed for milligram quantities would be
appropriate.
Another important consideration is the amount of ash that is formed during
the combustion and its removal. The ash will comprise the remains of tin and
aluminium containers and the inorganic residues from the test portion.
Instruments are manufactured with both vertical and horizontal furnace
configurations. With vertical systems, ceramic crucibles are placed in the
combustion tubes to accommodate the ash. This allows extended auto-sampler
operation but can lead to back-pressure problems if the ash is not removed on a
regular basis. In horizontal systems, the ash is removed after each combustion
although this arrangement is difficult to automate.
Applications of CHNS Elemental Analysers
CHNS elemental analysers have been used in analytical laboratories for
over thirty years. The method is used extensively across a wide range of
applications, including pharmaceuticals, chemicals, oil-related products, catalysts
and food. In the oil industry, an important application is the regular monitoring of
coke build-up on refinery catalysts to ensure that regeneration procedures
(involving controlled burning of the carbon) are executed at optimal intervals.
Since many of these catalyst systems involve large quantities of noble metals
such as platinum, palladium and rhenium, mismanagement of this testing would
entail serious financial losses. In food analysis, the determination of nitrogen (as
a surrogate for protein) is very important for pricing grain and evaluating meat
products, and is increasingly undertaken by combustion analysis.
ANALYSIS OF:
Carbon and hydrogen:

60
 • The sample is subjected to total oxidation, the carbon and hydrogen
converted into CO2 and water. From the weights of CO2 and H2O the %
of C n H in the sample are calculated.
• This analysis are carried on a microscale with a few milligrams of sample.
• The apparatus consists of a combustion tube to contain the sample and
appropriate catalysts, a furnace, a source of O2 with suitable pressure
controlling equipment and absorption tube
• The combustion tube contains silver, platinum, copper oxide, lead
chromate and peroxide.
• The sample is heated in the presence of these substances in a stream of
O2.
• The silver removes halogens and sulfur oxides from the vaporized sample.
• The platinum aids in the complete combustion of some resistant
compounds.
• The copper oxide- lead chromate mixture is an oxidizing agent.
• The lead peroxide removes oxides of nitrogen from the vapors.
• As the combusted vapors consists of CO2 n H2O, leave the combustion
tube, they are passed into an absorption tube of magnesium perchlorate
which removes water, this vapors then passed into a tube of NaoH
absorbed on asbestos, which removes all CO2.
• The absorption tubes are weighed before and after the combustion, thus
providing the weights of CO2 and H2O produced by the sample.
• The acceptable accuracy of the method is ±0.3% of the theoretical % of C
and H
Halogens:
• Analysis of halide content of halide and hydrohalide salts provides a rapid
indication of purity.
• Ex. It is inadvisable to analyze ephedrine Hcl merely by titrating the
chloride with silver nitrate, because the sample may contain another
chloride salt.
• Carius method: The halogen compound is sealed in a combustion tube
with silver nitrate and nitric acid and the tube is heated for several hours.
The organic material is decomposed
• Organic X → H2O + CO2 + X2
X2 → 2 AgX
The tube is opened and the insoluble silver halide is weighed
• Schoniger oxygen-flask combustion technique:
• It is simple, rapid method for halogen analysis.
61
 • The organic compound is wrapped in a filter paper and clamped in
platinum guaze.
• The assembly is liighted and immediately placed in a closed with filed
with O2.
• After the combustion is complete the gases are absorbed in a suitable
liquid, and this solution is analyzed.
• Parr method:
• Halogens are converted into their sodium salts, which can be analyzed by
conventional methods.
• Sulfur and phosphorus can also be determined by the carius, schoniger,
and parr methods.
• Sulfate is determined by titration with barium ion.
• Phosphate is determined by gravimetrically
STABILITY TESTING PROTOCOLS
INTRODUCTION
This guidance is the second revision of Q1A Stability Testing of New Drug
Substances and Products, which was first published in September 1994 and
revised in August 2001. The purpose of this revision is to harmonize the
intermediate storage condition for zones I and II with the long-term condition for
zones III and IV recommended in the ICH guidance Q1F Stability Data Package
for Registration Applications in Climatic Zones III and IV. The changes made in
this second revision are listed in the attachment to this guidance.
A. Objectives of the Guidance
This guidance is intended to define what stability data package for a new
drug substance or drug product is sufficient for a registration application within
the three regions of the European Union (EU), Japan, and the United States. It
does not seek to address the testing for registration in or export to other areas of
the world. The guidance exemplifies the core stability data package fornew drug
substances and products, but leaves sufficient flexibility to encompass the variety
ofdifferent practical situations that may be encountered due to specific scientific
considerations and characteristics of the materials being evaluated. Alternative
approaches can be used when there are scientifically justifiable reasons.
B. Scope of the Guidance
The guidance addresses the information to be submitted in registration
applications for new molecular entities and associated drug products. This

62
guidance does not currently seek to cover the information to be submitted for
abbreviated or abridged applications, variations, or clinical trial applications.
Specific details of the sampling and testing for particular dosage forms in
their proposed container closures are not covered in this guidance.
Further guidance on new dosage forms and on biotechnological/biological
products can be found in ICH guidances Q1C Stability Testing for New Dosage
Forms and Q5C Quality of Biotechnological Products: Stability Testing of
Biotechnological/Biological Products, respectively.
C. General Principles
The purpose of stability testing is to provide evidence on how the quality of
a drug substance or drug product varies with time under the influence of a variety
of environmental factors, such as temperature, humidity, and light, and to
establish a retest period for the drug substance or a shelf life for the drug product
and recommended storage conditions.
The choice of test conditions defined in this guidance is based on an
analysis of the effects of climatic conditions in the three regions of the EU,
Japan, and the United States. The mean kinetic temperature in any part of the
world can be derived from climatic data, and the world can be divided into four
climatic zones, I-IV.
This guidance addresses climatic zones I and II. The principle has been
established that stability information generated in any one of the three regions of
the EU, Japan, and the United States would be mutually acceptable to the other
two regions, provided the information is consistent with this guidance and the
labeling is in accord with national/regional requirements. FDA's guidance
documents, including this guidance, do not establish legally enforceable
responsibilities. Instead, guidances describe the Agency's current thinking on a
topic and should be viewed only as recommendations, unless specific regulatory
or statutory requirements are cited. The use of the word should in Agency
guidances means that something is suggested or recommended, but not required.
GUIDANCE
A. Drug Substance
1. General

63
 Information on the stability of the drug substance is an integral part of the
systematic approach to stability evaluation.
2. Stress Testing
Stress testing of the drug substance can help identify the likely degradation
products, which can in turn help establish the degradation pathways and the
intrinsic stability of the molecule and validate the stability indicating power of
the analytical procedures used. The nature of the stress testing will depend on the
individual drug substance and the type of drug product involved.
Stress testing is likely to be carried out on a single batch of the drug
substance. The testing should include the effect of temperatures (in 10°C
increments (e.g., 50°C, 60°C) above that for accelerated testing), humidity (e.g.,
75 percent relative humidity or greater) where appropriate, oxidation, and
photolysis on the drug substance. The testing should also evaluate the
susceptibility of the drug substance to hydrolysis across a wide range of pH
values when in solution or suspension. Photostability testing should be an
integral part of stress testing. The standard conditions for photostability testing
are described in ICH Q1B Photostability Testing of New Drug Substances and
Products.
Examining degradation products under stress conditions is useful in
establishing degradation pathways and developing and validating suitable
analytical procedures. However, such examination may not be necessary for
certain degradation products if it has been demonstrated that they are not formed
under accelerated or long-term storage conditions.
Results from these studies will form an integral part of the information
provided to regulatory authorities.
3. Selection of Batches
Data from formal stability studies should be provided on at least three
primary batches of the drug substance. The batches should be manufactured to a
minimum of pilot scale by the same synthetic route as production batches and
using a method of manufacture and procedure that simulates the final process to
be used for production batches. The overall quality of the batches of drug
substance placed on formal stability studies should be representative of the
quality of the material to be made on a production scale.
Other supporting data can be provided.
64
4. Container Closure System
The stability studies should be conducted on the drug substance packaged
in a container closure system that is the same as or simulates the packaging
proposed for storage and distribution.
5. Specification
Specification, which is a list of tests, references to analytical procedures,
and proposed acceptance criteria, is addressed in ICH Q6A Specifications: Test
Procedures and Acceptance Criteria for New Drug Substances and New Drug
Products: Chemical Substances and Q6B
Specifications: Test Procedures and Acceptance Criteria for New Drug
Substances and New Drug Products: Biotechnological/Biological Products. In
addition, specification for degradation products in a drug substance is discussed
in ICH Q3A Impurities in New Drug Substances.
Stability studies should include testing of those attributes of the drug
substance that are susceptible to change during storage and are likely to influence
quality, safety, and/or efficacy.
The testing should cover, as appropriate, the physical, chemical, biological,
and microbiological attributes. Validated stability-indicating analytical
procedures should be applied. Whether and to what extent replication should be
performed should depend on the results from validation studies.
6. Testing Frequency
For long-term studies, frequency of testing should be sufficient to establish
the stability profile of the drug substance. For drug substances with a proposed
retest period of at least 12 months, the frequency of testing at the long-term
storage condition should normally be every 3 months over the first year, every 6
months over the second year, and annually thereafter through the proposed retest
period.
At the accelerated storage condition, a minimum of three time points,
including the initial and final time points (e.g., 0, 3, and 6 months), from a 6-
month study is recommended. Where an expectation (based on development
experience) exists that the results from accelerated studies are likely to approach
significant change criteria, increased testing should be conducted either by

65
adding samples at the final time point or including a fourth time point in the
study design.
When testing at the intermediate storage condition is called for as a result
of significant change at the accelerated storage condition, a minimum of four
time points, including the initial and final time points (e.g., 0, 6, 9, 12 months),
from a 12-month study is recommended.
7. Storage Conditions
In general, a drug substance should be evaluated under storage conditions
(with appropriate tolerances) that test its thermal stability and, if applicable, its
sensitivity to moisture. The storage conditions and the lengths of studies chosen
should be sufficient to cover storage, shipment, and subsequent use.
The long-term testing should cover a minimum of 12 months’ duration on
at least three primary batches at the time of submission and should be continued
for a period of time sufficient to cover the proposed retest period. Additional data
accumulated during the assessment period of the registration application should
be submitted to the authorities if requested. Data from the accelerated storage
condition and, if appropriate, from the intermediate storage condition can be used
to evaluate the effect of short-term excursions outside the label storage
conditions (such as might occur during shipping).
Long-term, accelerated, and, where appropriate, intermediate storage
conditions for drug substances are detailed in the sections below. The general
case should apply if the drug substance is not specifically covered by a
subsequent section. Alternative storage conditions can be used if justified.
a. General case

66
 If long-term studies are conducted at 25°C ± 2°C/60% RH ± 5% RH and
significant change occurs at any time during 6 months’ testing at the accelerated
storage condition, additional testing at the intermediate storage condition should
be conducted and evaluated against significant change criteria.
Testing at the intermediate storage condition should include all tests, unless
otherwise justified. The initial application should include a minimum of 6
months’ data from a 12-month study at the intermediate storage condition.
Significant change for a drug substance is defined as failure to meet its
specification.

b. Drug substances intended for storage in a refrigerator

Data from refrigerated storage should be assessed according to the
evaluation section of this guidance, except where explicitly noted below.
If significant change occurs between 3 and 6 months’ testing at the
accelerated storage condition, the proposed retest period should be based on the
real time data available at the long-term storage condition.
If significant change occurs within the first 3 months’ testing at the
accelerated storage condition, a discussion should be provided to address the

67
effect of short-term excursions outside the label storage condition (e.g., during
shipping or handling). This discussion can be supported, if appropriate, by
further testing on a single batch of the drug substance for a period shorter than
3 months but with more frequent testing than usual. It is considered
unnecessary to continue to test a drug substance through 6 months when a
significant change has occurred within the first 3 months.
c. Drug substances intended for storage in a freezer

For drug substances intended for storage in a freezer, the retest period
should be based on the real time data obtained at the long-term storage condition.
In the absence of an accelerated storage condition for drug substances intended to
be stored in a freezer, testing on a single batch at an elevated temperature (e.g.,
5°C ± 3°C or 25°C ± 2°C) for an appropriate time period should be conducted to
address the effect of short-term excursions outside the proposed label storage
condition (e.g., during shipping or handling).
d. Drug substances intended for storage below -20°C
Drug substances intended for storage below -20°C should be treated on a
case-by-case basis.
8. Stability Commitment
When available long-term stability data on primary batches do not cover
the proposed retest period granted at the time of approval, a commitment should
be made to continue the stability studies postapproval to firmly establish the
retest period. Where the submission includes long-term stability data on three
production batches covering the proposed retest period, a postapproval
commitment is considered unnecessary. Otherwise, one of the following
commitments should be made:
 If the submission includes data from stability studies on at least three
production batches, a commitment should be made to continue these studies
through the proposed retest period.
 If the submission includes data from stability studies on fewer than three
production batches, a commitment should be made to continue these studies
through the proposed retest period and to place additional production
68
 batches, to a total of at least three, on long-term stability studies through the
proposed retest
period.
 If the submission does not include stability data on production batches, a
commitment should be made to place the first three production batches on
longterm
stability studies through the proposed retest period.
The stability protocol used for long-term studies for the stability
commitment should be the same as that for the primary batches, unless otherwise
scientifically justified.
9. Evaluation
The purpose of the stability study is to establish, based on testing a
minimum of three batches of the drug substance and evaluating the stability
information (including, as appropriate, results of the physical, chemical,
biological, and microbiological tests), a retest period applicable to all future
batches of the drug substance manufactured under similar circumstances.
The degree of variability of individual batches affects the confidence that a
future production batch will remain within specification throughout the assigned
retest period.
The data may show so little degradation and so little variability that it is
apparent from looking at the data that the requested retest period will be granted.
Under these circumstances, it is normally unnecessary to go through the formal
statistical analysis; providing a justification for the omission should be sufficient.
An approach for analyzing the data on a quantitative attribute that is
expected to change with time is to determine the time at which the 95 percent,
one-sided confidence limit for the mean curve intersects the acceptance criterion.
If analysis shows that the batch-to-batch variability is small, it is advantageous to
combine the data into one overall estimate. This can be done by first applying
appropriate statistical tests (e.g., p values for level of significance of rejection of
more than 0.25) to the slopes of the regression lines and zero time intercepts for
the individual batches.
If it is inappropriate to combine data from several batches, the overall retest
period should be based on the minimum time a batch can be expected to remain
within acceptance criteria.
69
 The nature of any degradation relationship will determine whether the data
should be transformed for linear regression analysis. Usually the relationship can
be represented by a linear, quadratic, or cubic function on an arithmetic or
logarithmic scale. Statistical methods should be employed to test the goodness of
fit of the data on all batches and combined batches (where appropriate) to the
assumed degradation line or curve.
Limited extrapolation of the real time data from the long-term storage
condition beyond the observed range to extend the retest period can be
undertaken at approval time if justified. This justification should be based, for
example, on what is known about the mechanism of degradation, the results of
testing under accelerated conditions, the goodness of fit of any mathematical
model, batch size, and/or existence of supporting stability data. However, this
extrapolation assumes that the same degradation relationship will continue to
apply beyond the observed data.
Any evaluation should cover not only the assay, but also the levels of
degradation products and other appropriate attributes.
10. Statements/Labeling
A storage statement should be established for the labeling in accordance
with relevant national/regional requirements. The statement should be based on
the stability evaluation of the drug substance. Where applicable, specific
instructions should be provided, particularly for drug substances that cannot
tolerate freezing. Terms such as ambient conditions or room temperature should
be avoided.
A retest period should be derived from the stability information, and a
retest date should be displayed on the container label if appropriate.
B. Drug Product
1. General
The design of the formal stability studies for the drug product should be
based on knowledge of the behavior and properties of the drug substance, results
from stability studies on the drug substance, and experience gained from clinical
formulation studies. The likely changes on storage and the rationale for the
selection of attributes to be tested in the formal stability studies should be stated.
2. Photostability Testing
70
 Photostability testing should be conducted on at least one primary batch of
the drug product if appropriate. The standard conditions for photostability testing
are described in ICH Q1B.
3. Selection of Batches
Data from stability studies should be provided on at least three primary
batches of the drug product. The primary batches should be of the same
formulation and packaged in the same container closure system as proposed for
marketing. The manufacturing process used for primary batches should simulate
that to be applied to production batches and should provide product of the same
quality and meeting the same specification as that intended for marketing.
Two of the three batches should be at least pilot scale batches, and the third
one can be smaller if justified. Where possible, batches of the drug product
should be manufactured by using different batches of the drug substance.
Stability studies should be performed on each individual strength and
container size of the drug product unless bracketing or matrixing is applied.
Other supporting data can be provided.
4. Container Closure System
Stability testing should be conducted on the dosage form packaged in the
container closure system proposed for marketing (including, as appropriate, any
secondary packaging and container label). Any available studies carried out on
the drug product outside its immediate container or in other packaging materials
can form a useful part of the stress testing of the dosage form or can be
considered as supporting information, respectively.
5. Specification
Specification, which is a list of tests, references to analytical procedures,
and proposed acceptance criteria, including the concept of different acceptance
criteria for release and shelf life specifications, is addressed in ICH Q6A and
Q6B. In addition, specification for degradation products in a drug product is
addressed in ICH Q3B Impurities in New Drug Products.
Stability studies should include testing of those attributes of the drug
product that are susceptible to change during storage and are likely to influence
quality, safety, and/or efficacy. The testing should cover, as appropriate, the
physical, chemical, biological, and microbiological attributes, preservative
71
content (e.g., antioxidant, antimicrobial preservative), and functionality tests
(e.g., for a dose delivery system). Analytical procedures should be fully validated
and stability indicating. Whether and to what extent replication should be
performed will depend on the results of validation studies.
Shelf life acceptance criteria should be derived from consideration of all
available stability information. It may be appropriate to have justifiable
differences between the shelf life and release acceptance criteria based on the
stability evaluation and the changes observed on storage. Any differences
between the release and shelf life acceptance criteria for antimicrobial
preservative content should be supported by a validated correlation of chemical
content and preservative effectiveness demonstrated during drug development on
the product in its final formulation (except for preservative concentration)
intended for marketing. A single primary stability batch of the drug product
should be tested for antimicrobial preservative effectiveness (in addition to
preservative content) at the proposed shelf life for verification purposes,
regardless of whether there is a difference between the release and shelf life
acceptance criteria for preservative content.
6. Testing Frequency
For long-term studies, frequency of testing should be sufficient to establish
the stability profile of the drug product. For products with a proposed shelf life of
at least 12 months, the frequency of testing at the long-term storage condition
should normally be every 3 months over the first year, every 6 months over the
second year, and annually thereafter through the proposed shelf life.
At the accelerated storage condition, a minimum of three time points,
including the initial and final time points (e.g., 0, 3, and 6 months), from a 6-
month study is recommended. Where an expectation (based on development
experience) exists that results from accelerated testing are likely to approach
significant change criteria, increased testing should be conducted either by
adding samples at the final time point or by including a fourth time point in the
study design.
When testing at the intermediate storage condition is called for as a result
of significant change at the accelerated storage condition, a minimum of four
time points, including the initial and final time points (e.g., 0, 6, 9, 12 months),
from a 12-month study is recommended. Reduced designs (i.e., matrixing or

72
bracketing), where the testing frequency is reduced or certain factor
combinations are not tested at all, can be applied if justified.
7. Storage Conditions
In general, a drug product should be evaluated under storage conditions
(with appropriate tolerances) that test its thermal stability and, if applicable, its
sensitivity to moisture or potential for solvent loss. The storage conditions and
the lengths of studies chosen should be sufficient to cover storage, shipment, and
subsequent use.
Stability testing of the drug product after constitution or dilution, if
applicable, should be conducted to provide information for the labeling on the
preparation, storage condition, and inuse period of the constituted or diluted
product. This testing should be performed on the constituted or diluted product
through the proposed in-use period on primary batches as part of the formal
stability studies at initial and final time points, and if full shelf life, long-term
data will not be available before submission, at 12 months or the last time point
for which data will be available. In general, this testing need not be repeated on
commitment batches.
The long-term testing should cover a minimum of 12 months’ duration on
at least three primary batches at the time of submission and should be continued
for a period of time sufficient to cover the proposed shelf life. Additional data
accumulated during the assessment period of the registration application should
be submitted to the authorities if requested. Data from the accelerated storage
condition and, if appropriate, from the intermediate storage condition can be used
to evaluate the effect of short-term excursions outside the label storage
conditions (such as might occur during shipping).
Long-term, accelerated, and, where appropriate, intermediate storage
conditions for drug products are detailed in the sections below. The general case
should apply if the drug product is not specifically covered by a subsequent
section. Alternative storage conditions can be used if justified.

73
a. General case
If long-term studies are condcuted at 25°C ± 2°C/60% RH ± 5% RH and
significant change occurs at any time during 6 months’ testing at the accelerated
storage condition, additional testing at the intermediate storage condition should
be conducted and evaluated against significant change criteria. The initial
application should include a minimum of 6 months’ data from a 12-month study
at the intermediate storage condition.
In general, significant change for a drug product is defined as one or more
of the following (as appropriate for the dosage form):
 A 5 percent change in assay from its initial value, or failure to meet the
acceptance criteria for potency when using biological or immunological
procedures
 Any degradation product’s exceeding its acceptance criterion
 Failure to meet the acceptance criteria for appearance, physical attributes,
and functionality test (e.g., color, phase separation, resuspendibility,
caking, hardness, dose delivery per actuation). However, some changes in
physical attributes (e.g., softening of suppositories, melting of creams) may
be expected under accelerated conditions.
 Failure to meet the acceptance criterion for pH
 Failure to meet the acceptance criteria for dissolution for 12 dosage units
b. Drug products packaged in impermeable containers
Sensitivity to moisture or potential for solvent loss is not a concern for drug
products packaged in impermeable containers that provide a permanent barrier to
passage of moisture or solvent.
Thus, stability studies for products stored in impermeable containers can be
conducted under any controlled or ambient humidity condition.
74
c. Drug products packaged in semipermeable containers
Aqueous-based products packaged in semipermeable containers should be
evaluated for potential water loss in addition to physical, chemical, biological,
and microbiological stability. This evaluation can be carried out under conditions
of low relative humidity, as discussed below.
Ultimately, it should be demonstrated that aqueous-based drug products
stored in semipermeable containers can withstand low relative humidity
environments. Other comparable approaches can be developed and reported for
nonaqueous, solvent-based products.

When long-term studies are conducted at 25°C ± 2°C/40% RH ± 5% RH

and significant change other than water loss occurs during the 6 months’ testing
at the accelerated storage condition, additional testing at the intermediate storage
condition should be performed, as described under the general case, to evaluate
the temperature effect at 30°C. A significant change in water loss alone at the
accelerated storage condition does not necessitate testing at the intermediate
storage condition. However, data should be provided to demonstrate that the drug
product will not have significant water loss throughout the proposed shelf life if
stored at 25°C and the reference relative humidity of 40 percent RH.
A 5 percent loss in water from its initial value is considered a significant
change for a product packaged in a semipermeable container after an equivalent
of 3 months’ storage at 40°C/NMT 25 percent RH. However, for small
containers (1 mL or less) or unit-dose products, a water loss of 5 percent or more
after an equivalent of 3 months’ storage at 40°C/NMT 25 percent RH may be
appropriate if justified.

75
 An alternative approach to studying at the reference relative humidity as
recommended in the table above (for either long-term or accelerated testing) is
performing the stability studies under higher relative humidity and deriving the
water loss at the reference relative humidity through calculation. This can be
achieved by experimentally determining the permeation coefficient for the
container closure system or, as shown in the example below, using the calculated
ratio of water loss rates between the two humidity conditions at the same
temperature. The permeation coefficient for a container closure system can be
experimentally determined by using the worst case scenario (e.g., the most
diluted of a series of concentrations) for the proposed drug product.
Example of an approach for determining water loss:
For a product in a given container closure system, container size, and fill,
an appropriate approach for deriving the water loss rate at the reference relative
humidity is to multiply the water loss rate measured at an alternative relative
humidity at the same temperature by a water loss rate ratio shown in the table
below. A linear water loss rate at the alternative relative humidity over the
storage period should be demonstrated.
For example, at a given temperature (e.g., 40°C), the calculated water loss
rate during storage at NMT 25 percent RH is the water loss rate measured at 75

percent RH multiplied by 3.0, the corresponding water loss rate ratio.

Valid water loss rate ratios at relative humidity conditions other than those
shown in the table above can also be used.

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d. Drug products intended for storage in a refrigerator
If the drug product is packaged in a semipermeable container, appropriate
information should be provided to assess the extent of water loss.
Data from refrigerated storage should be assessed according to the
evaluation section of this guidance, except where explicitly noted below.
If significant change occurs between 3 and 6 months’ testing at the
accelerated storage condition, the proposed shelf life should be based on the real
time data available from the long-term storage condition.
If significant change occurs within the first 3 months’ testing at the
accelerated storage condition, a discussion should be provided to address the
effect of short-term excursions outside the label storage condition (e.g., during
shipment and handling). This discussion can be supported, if appropriate, by
further testing on a single batch of the drug product for a period shorter than 3
months but with more frequent testing than usual. It is considered unnecessary to
continue to test a product through 6 months when a significant change has
occurred within the first 3 months.

e. Drug products intended for storage in a freezer

For drug products intended for storage in a freezer, the shelf life should be
based on the real time data obtained at the long-term storage condition. In the
absence of an accelerated storage condition for drug products intended to be
stored in a freezer, testing on a single batch at an elevated temperature (e.g., 5°C
± 3°C or 25°C ± 2°C) for an appropriate time period should be conducted to
address the effect of short-term excursions outside the proposed label storage
condition.
77
f. Drug products intended for storage below -20°C
Drug products intended for storage below -20°C should be treated on a
case-by-case basis.
8. Stability Commitment
When available long-term stability data on primary batches do not cover
the proposed shelf life granted at the time of approval, a commitment should be
made to continue the stability studies postapproval to firmly establish the shelf
life. Where the submission includes long-term stability data from three
production batches covering the proposed shelf life, a postapproval commitment
is considered unnecessary. Otherwise, one of the following commitments should
be made:
 If the submission includes data from stability studies on at least three
production batches, a commitment should be made to continue the long-
term studies through the proposed shelf life and the accelerated studies for
6 months.
 If the submission includes data from stability studies on fewer than three
production batches, a commitment should be made to continue the long-
term studies through the proposed shelf life and the accelerated studies for
6 months, and to place additional production batches, to a total of at least
three, on long-term stability studies through the proposed shelf life and on
accelerate studies for 6 months.
 If the submission does not include stability data on production batches, a
commitment should be made to place the first three production batches on
longterm stability studies through the proposed shelf life and on accelerated
studies for 6 months. The stability protocol used for studies on commitment
batches should be the same as that for the primary batches, unless otherwise
scientifically justified. Where intermediate testing is called for by a
significant change at the accelerated storage condition for the primary
batches, testing on the commitment batches can be conducted at either the
intermediate or the accelerated storage condition. However, if significant
change occurs at the accelerated storage condition on the commitment
batches, testing at the intermediate storage condition should also be
conducted.
9. Evaluation
A systematic approach should be adopted in the presentation and evaluation
of the stability information, which should include, as appropriate, results from

78
the physical, chemical, biological, and microbiological tests, including particular
attributes of the dosage form (e.g., dissolution rate for solid oral dosage forms).
The purpose of the stability study is to establish, based on testing a
minimum of three batches of the drug product, a shelf life and label storage
instructions applicable to all future batches of the drug product manufactured and
packaged under similar circumstances. The degree of variability of individual
batches affects the confidence that a future production batch will remain within
specification throughout its shelf life.
Where the data show so little degradation and so little variability that it is
apparent from looking at the data that the requested shelf life will be granted, it is
normally unnecessary to go through the formal statistical analysis; providing a
justification for the omission should be sufficient. An approach for analyzing
data of a quantitative attribute that is expected to change with time is to
determine the time at which the 95 percent one-sided confidence limit for the
mean curve intersects the acceptance criterion. If analysis shows that the batch-
to-batch variability is small, it is advantageous to combine the data into one
overall estimate. This can be done by first applying appropriate statistical tests
(e.g., p values for level of significance of rejection of more than 0.25) to the
slopes of the regression lines and zero time intercepts for the individual batches.
If it is inappropriate to combine data from several batches, the overall shelf
life should be based on the minimum time a batch can be expected to remain
within acceptance criteria.
The nature of the degradation relationship will determine whether the data
should be transformed for linear regression analysis. Usually the relationship can
be represented by a linear, quadratic, or cubic function on an arithmetic or
logarithmic scale. Statistical methods should be employed to test the goodness of
fit on all batches and combined batches (where appropriate) to the assumed
degradation line or curve.
Limited extrapolation of the real time data from the long-term storage
condition beyond the observed range to extend the shelf life can be undertaken at
approval time if justified. This justification should be based, for example, on
what is known about the mechanisms of degradation, the results of testing under
accelerated conditions, the goodness of fit of any mathematical model, batch
size, and/or existence of supporting stability data. However, this extrapolation

79
assumes that the same degradation relationship will continue to apply beyond the
observed data.
Any evaluation should consider not only the assay but also the degradation
products and other appropriate attributes. Where appropriate, attention should be
paid to reviewing the adequacy of the mass balance and different stability and
degradation performance.
10. Statements/Labeling
A storage statement should be established for the labeling in accordance
with relevant national/regional requirements. The statement should be based on
the stability evaluation of the drug product. Where applicable, specific
instruction should be provided, particularly for drug products that cannot tolerate
freezing. Terms such as ambient conditions or room temperature should be
avoided.
There should be a direct link between the label storage statement and the
demonstrated stability of the drug product. An expiration date should be
displayed on the container label.
GLOSSARY
The following definitions are provided to facilitate interpretation of the
guidance.
Accelerated testing:
Studies designed to increase the rate of chemical degradation or physical
change of a drug substance or drug product by using exaggerated storage
conditions as part of the formal stability studies. Data from these studies, in
addition to long-term stability studies, can be used to assess longer term chemical
effects at nonaccelerated conditions and to evaluate the effect of short-term
excursions outside the label storage conditions such as might occur during
shipping. Results from accelerated testing studies are not always predictive of
physical changes.
Bracketing:
The design of a stability schedule such that only samples on the extremes of
certain design factors (e.g., strength, package size) are tested at all time points as
in a full design. The design assumes that the stability of any intermediate levels is
represented by the stability of the extremes tested. Where a range of strengths is
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to be tested, bracketing is applicable if the strengths are identical or very closely
related in composition (e.g., for a tablet range made with different compression
weights of a similar basic granulation, or a capsule range made by filling
different plug fill weights of the same basic composition into different size
capsule shells). Bracketing can be applied to different container sizes or different
fills in the same container closure system.
Climatic zones:
The four zones in the world that are distinguished by their characteristic,
prevalent annual climatic conditions. This is based on the concept described by
W. Grimm (Drugs Made in Germany, 28:196-202, 1985 and 29:39-47, 1986).
Commitment batches:
Production batches of a drug substance or drug product for which the
stability studies are initiated or completed postapproval through a commitment
made in the registration application.
Container closure system:
The sum of packaging components that together contain and protect the
dosage form. This includes primary packaging components and secondary
packaging components if the latter are intended to provide additional protection
to the drug product. A packaging system is equivalent to a container closure
system.
Dosage form:
A pharmaceutical product type (e.g., tablet, capsule, solution, cream) that
contains a drug substance generally, but not necessarily, in association with
excipients.
Drug product:
The dosage form in the final immediate packaging intended for marketing.
Drug substance:
The unformulated drug substance that may subsequently be formulated
with excipients to produce the dosage form.
Excipient:
Anything other than the drug substance in the dosage form.
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Expiration date:
The date placed on the container label of a drug product designating the
time prior to which a batch of the product is expected to remain within the
approved shelf life specification, if stored under defined conditions, and after
which it must not be used.
Formal stability studies:
Long-term and accelerated (and intermediate) studies undertaken on
primary and/or commitment batches according to a prescribed stability protocol
to establish or confirm the retest period of a drug substance or the shelf life of a
drug product.
Impermeable containers:
Containers that provide a permanent barrier to the passage of gases or
solvents (e.g., sealed aluminum tubes for semi-solids, sealed glass ampoules for
solutions).
Intermediate testing:
Studies conducted at 30°C/65% RH and designed to moderately increase
the rate of chemical degradation or physical changes for a drug substance or drug
product intended to be stored long-term at 25°C.
Long-term testing:
Stability studies under the recommended storage condition for the retest
period or shelf life proposed (or approved) for labeling.
Mass balance:
The process of adding together the assay value and levels of degradation
products to see how closely these add up to 100 percent of the initial value, with
due consideration of the margin of analytical error.
Matrixing:
The design of a stability schedule such that a selected subset of the total
number of possible samples for all factor combinations is tested at a specified
time point. At a subsequent time point, another subset of samples for all factor
combinations is tested. The design assumes that the stability of each subset of
samples tested represents the stability of all samples at a given time point. The
differences in the samples for the same drug product should be identified as, for
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example, covering different batches, different strengths, different sizes of the
same container closure system, and, possibly in some cases, different container
closure systems.
Mean kinetic temperature:
A single derived temperature that, if maintained over a defined period of
time, affords the same thermal challenge to a drug substance or drug product as
would be experienced over a range of both higher and lower temperatures for an
equivalent defined period. The mean kinetic temperature is higher than the
arithmetic mean temperature and takes into account the Arrhenius equation.
When establishing the mean kinetic temperature for a defined period, the formula
of J. D. Haynes (J. Pharm. Sci., 60:927-929, 1971) can be used.
New molecular entity:
An active pharmaceutical substance not previously contained in any drug
product registered with the national or regional authority concerned. A new salt,
ester, or noncovalent bond derivative of an approved drug substance is
considered a new molecular entity for the purpose of stability testing under this
guidance.
Pilot scale batch:
A batch of a drug substance or drug product manufactured by a procedure
fully representative of and simulating that to be applied to a full production scale
batch. For solid oral dosage forms, a pilot scale is generally, at a minimum, one-
tenth that of a full production scale or 100,000 tablets or capsules, whichever is
larger.
Primary batch:
A batch of a drug substance or drug product used in a formal stability
study, from which stability data are submitted in a registration application for the
purpose of establishing a retest period or shelf life, respectively. A primary batch
of a drug substance should be at least a pilot scale batch. For a drug product, two
of the three batches should be at least pilot scale batch, and the third batch can be
smaller if it is representative with regard to the critical manufacturing steps.
However, a primary batch may be a production batch.
Production batch:

83
 A batch of a drug substance or drug product manufactured at production
scale by using production equipment in a production facility as specified in the
application.
Retest date:
The date after which samples of the drug substance should be examined to
ensure that the material is still in compliance with the specification and thus
suitable for use in the manufacture of a given drug product.
Retest period:
The period of time during which the drug substance is expected to remain
within its specification and, therefore, can be used in the manufacture of a given
drug product, provided that the drug substance has been stored under the defined
conditions. After this period, a batch of drug substance destined for use in the
manufacture of a drug product should be retested for compliance with the
specification and then used immediately. A batch of drug substance can be
retested multiple times and a different portion of the batch used after each retest,
as long as it continues to comply with the specification. For most
biotechnological/biological substances known to be labile, it is more appropriate
to establish a shelf life than a retest period. The same may be true for certain
antibiotics.
Semipermeable containers:
Containers that allow the passage of solvent, usually water, while
preventing solute loss. The mechanism for solvent transport occurs by absorption
into one container surface, diffusion through the bulk of the container material,
and desorption from the other surface. Transport is driven by a partial pressure
gradient. Examples of semipermeable containers include plastic bags and
semirigid, low-density polyethylene (LDPE) pouches for large volume
parenterals (LVPs), and LDPE ampoules, bottles, and vials.
Shelf life (also referred to as expiration dating period):
The time period during which a drug product is expected to remain within
the approved shelf life specification, provided that it is stored under the
conditions defined on the container label.
Specification:
See ICH Q6A and Q6B.
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Specification, Release:
The combination of physical, chemical, biological, and microbiological
tests and acceptance criteria that determine the suitability of a drug product at the
time of its release.
Specification, Shelf life:
The combination of physical, chemical, biological, and microbiological
tests and acceptance criteria that determine the suitability of a drug substance
throughout its retest period, or that a drug product should meet throughout its
shelf life.
Storage condition tolerances:
The acceptable variations in temperature and relative humidity of storage
facilities for formal stability studies. The equipment should be capable of
controlling the storage condition within the ranges defined in this guidance. The
actual temperature and humidity (when controlled) should be monitored during
stability storage. Short-term spikes due to opening of doors of the storage facility
are accepted as unavoidable. The effect of excursions due to equipment failure
should be addressed and reported if judged to affect stability results. Excursions
that exceed the defined tolerances for more than 24 hours should be described in
the study report and their effect assessed.
Stress testing (drug substance):
Studies undertaken to elucidate the intrinsic stability of the drug substance.
Such testing is part of the development strategy and is normally carried out under
more severe conditions than those used for accelerated testing.
Stress testing (drug product):
Studies undertaken to assess the effect of severe conditions on the drug
product. Such studies include photostability testing (see ICH Q1B) and specific
testing of certain products (e.g., metered dose inhalers, creams, emulsions,
refrigerated aqueous liquid products).
Supporting data:
Data, other than those from formal stability studies, that support the
analytical procedures, the proposed retest period or shelf life, and the label
storage statements. Such data include (1) stability data on early synthetic route
batches of drug substance, small scale batches of materials, investigational
85
formulations not proposed for marketing, related formulations, and product
presented in containers and closures other than those proposed for marketing; (2)
information regarding test results on containers; and (3) other scientific
rationales.
STABILITY
The term “stability,” with respect to a drug dosage form, refers to the
chemical and physical integrity of the dosage unit and, when appropriate, the
ability of the dosage unit to maintain protection against microbiological
contamination. The shelf life of the dosage form is the time lapse from initial
preparation to the specified expiration date. The monograph specifications of
identity, strength, quality, and purity apply throughout the shelf life of the
product. The stability of the product is its ability to resist deterioration. It is
always expressed in terms of shelf life. Stability is the capacity of a drug product
to remain within specifications established to ensure its identity, strength quality
and purity. (USP-NF)
The stability parameters of a drug dosage form can be influenced by
environmental conditions of storage (temperature, light, air, and humidity), as
well as the package components. Pharmacopeial articles should include required
storage conditions on their labeling. These are the conditions under which the
expiration date shall apply. The storage requirements specified in the labeling for
the article must be observed throughout the distribution of the article (i.e.,
beyond the time it leaves the manufacturer up to and including its handling by
the dispenser or seller of the article to the consumer). Although labeling for the
consumer should indicate proper storage conditions, it is recognized that control
beyond the dispenser or seller is difficult. The beyond-use date shall be placed on
the container label.

Stability Protocols
Stability of manufactured dosage forms must be demonstrated by the
manufacturer, using methods adequate for the purpose. Monograph assays may
be used for stability testing if they are stability-indicating (i.e., if they accurately
differentiate between the intact drug molecules and their degradation products).
Stability considerations should include not only the specific compendial
requirements, but also changes in physical appearance of the product that would
warn users that the product's continued integrity is questionable.
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 Stability studies on active substances and packaged dosage forms are
conducted by means of “real-time,” long-term tests at specific temperatures and
relative humidities representing storage conditions experienced in the distribution
chain of the climatic zone(s) of the country or region of the world concerned.
Labeling of the packaged active substance or dosage form should reflect the
effects of temperature, relative humidity, air, and light on its stability. Label
temperature storage warnings will both reflect the results of the real-time storage
tests and allow for expected seasonal excursions of temperature

Guidelines for Pharmaceutical Stability Study : Pharmaceutical

Guidelines
Following are the guidelines for stability study conduction for new products:

1. Formal stability study should consist of accelerated and long term

stability testing on at least two primary production batches for stable drug
products and in case of the susceptible drug products at least three primary
production batches should be considered.

2. The accelerated stability testing data at 40°C / 75% for minimum six
months and long term stability testing data at 30°C / 65% for minimum 12
months should be available at the time of submission for new drug application
and can be continued further..

3. The product stable for 6 months at 40°C / 75% (Accelerated stability

conditions) then it can be assigned the shelf life of 24 months.

4. If the shelf life period exceeding the 24 months is to be assigned for the
product the real time stability data should be available.

5. Though not accepted internationally, as internal policy decision we can

give the shelf life of 36 months if product is found stable at accelerated stability
conditions of 40°C / 75% for 12 months.

6. The shelf life of 36 months or more can be assigned to the drug

formulation after completion of long term stability study for 36 months or more.

7. If there is change in the primary packing material the product should be

treated as new product for conduction of stability studies.

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 8. The stability studies should be performed on each individual strength of
the drug product unless bracketing is applied.

9. If the same product is having the different doses (different strengths) and
identical production formulation, and but different production process then each
should be treated as new product the stability study should be carried out
separately for each of the strengths.

10. The frequency of the testing for long term stability testing should be
initial and after every 3 months over the first year, every 6 months over second
year and annually thereafter through out the proposed shelf life.

11. The frequency of the testing for accelerated stability testing should be
initial 3 months and 6 months.

12. While labeling the stability samples the terms ambient conditions or
room temperature are not acceptable.

13. The stability testing should cover chemical, physical, biological and
microbiological attributes including preservative content and the testing of those
attributes of the drug products that are susceptible to change during storage and
are likely to influence quality, safety and or efficacy of the drug product.

14. Out of three batches selected for stability study testing, the at least two
batches should be pilot scale batches and third one can be smaller if justified.

COMMITMENT
Commitment may be defined as the state or quality of being dedicated to a
cause, activity, etc.
Professional Commitment

The principal goal of pharmaceutical care is to achieve positive outcomes

from the use of medication which improves patients’ quality of life with
minimum risk. Pharmacists strive to:

 Cure disease;
 Eliminate or reduce symptoms;
 Arrest or slow a disease process;
 Prevent disease; Diagnose disease; and
 Alter physiological processes for desirable result in the patient’s
health.
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 Pharmacists are professionals, uniquely prepared and available,
committed to public service and to the achievement of this goal.

STABILITY STUDY COMMITMENTS

Initial Study Commitments
a. Purpose
Since stability data submitted as part of a new animal drug application are
almost invariably based on laboratory or pilot-scale batches, the problems
associated with the scale-up to full-size production batches must be considered.
In order to demonstrate that the transition can be made from pilot-scale to full-
scale production without encountering stability problems, a stability-testing
protocol with corresponding commitments should be developed and submitted.
b. Protocol and Commitments
The protocol for a stability study with necessary commitments should
include the following information:
i. The number and/or frequency of production batches to be placed in a
continuing stability study.
ii. Description of the chemical, microbiological, and physical tests to be
used to monitor the stability of the drug product.
iii. Description of the containers to be used.
iv. The proposed environmental and storage conditions under which the
drug product will be studied and then marketed.
v. Frequency or intervals of testing.
vi. Frequency at which results will be reported.
vii. Provisions to test batches manufactured immediately before and after a
production batch which is found to be out-of-specifications.
viii. Provisions for withdrawal from the market of any production batch
found to be out-of-specifications and, if necessary, those batches immediately
before and after the batch in question.
Product/Container Changes/Alternate Drug Substance Suppliers

89
 Whenever changes in formulation (changes in drug concentration or
inactive ingredients), use of new containers, alternate sources, etc., are made, the
product manufactured under the new conditions should be placed into the
ongoing approved stability study.
A commitment should specify all conditions relative to the new change and
a statement to perform any additional studies needed for the changes in the
application.
In most cases, the approved expiration date may be used. However, this
will be determined at the time of the review of the supplemental submission and
requested action.
Analysis of Distribution Samples
Although not mandatory, it may be desirable to obtain samples from
distributed batches for stability testing. Ideally, these samples should obtained
from geographical areas which represent extremes of temperature and humidity.
It should be noted that the above voluntary activity does not relieve the
applicant from the requirements for stability testing as established under cGMPs.
Post-Approval Studies
After approval, the sponsor may make a request to liberalize or remove
storage condition restrictions, to adjust (extend, reduce or remove) an expiration
dating period, remove or provide a storage temperature restriction, provide for
use of a different container, or amend existing commitments. In these cases,
adequate stability data should be presented to justify the request.
STABILITY STUDY COMMITMENT FORMAT
A commitment to assure stability of a product should specifically define the
following items.
Samples of Testing
The first three (3) production lots should be tested followed by 3 to 10% of
annual production lots (with a minimum of 1 lot per year).
The number and/or frequency of production batches to be placed on a
continuing stability study should be based on a consideration of the anticipated
annual production and known stability profile.
Control Tests
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 The chemical, microbiological, and physical tests to be used to monitor the
stability of the drug product should be described.
Tests and methods must be appropriate and adequate for the study.
Assay methods must be stability-indicating, i.e., they must accurately
differentiate between the intact drug molecule(s), any possible degradation
products and product excipients. Methods must be adequately validated.
Container
The container to be used should be described.
The container and closure system should be those proposed for use in
marketing the product.
Product Storage Conditions
Product storage conditions must specify type of storage, temperature and
humidity conditions, light, etc. These conditions must be applicable to the drug
product in the market container and label requirements.
Frequency or Interval of Testing
The schedule of testing samples relative to the proposed expiration date
period at the recommended temperatures should be described.
Result Reporting
Prior to approval for marketing, all available data should be submitted with
the original applications and any amendments. Post-approval data may be
reported in the Drug Experience Report (DER) or in supplemental applications.
Withdrawal Provisions
Provisions to test batches manufactured immediately before and after a
batch which is found to be out-of-specifications should be provided.
Provisions for withdrawal from the market of any production batch found
to be out-of-specifications and, if necessary, those batches immediately before
and after the batch in question should be provided.
EFFECT OF FACTORS AFFECTING REACTION RATES ON
STABILITY
Ionic Strength (Primary Salt Effects )

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 For drug degradation involving reactions with or between ionic species, the
rate is affected by the presence of other ionic species such as salts like sodium
chloride. Ionic strength affects the observed degradation rate constant, k, by its
effect on the activityn coefficients, ƒ. Ionic strength, µ, is described by where Ci
is the concentration of ionic species i and Zi is its electric charge.
TEMPERATURE
It is one of the primary factors affecting drug stability. The rate
constant/temperature relationship has traditionally been described by the
Arhenius equation, k = A exp(-Ea/RT) where E a = activation energy A =
frequency factor .
REMEDIES :- Pharmaceutical product should be stored within the
temperature range in which they are stable. They should not be exposed to
extremes of temperature. Usually they should be stored at low temperature if they
lack sufficient stability at room temperature. There are few drugs on which
freezing has an adverse effect, so freezing should be avoided unless until it is
stable at such temperatures.
pH AND pH RATE PROFILES
Second most important parameter. The effect of pH on degradation rate can
be explained by the catalytic effects that hydronium or hydroxide ions can have
on various chemical reactions. If critical path in a reaction involves a proton
transfer or abstraction step, other acids and bases present in solution can affect
the rate of reaction.
A reaction in which hydronium ion, hydroxide ion, and water catalysis are
observed can be described by
K obs = k H+ a H+ + K H2O + K oH- a OH-
Where K obs = sum of specific rate constants
a H+ = activities of hydronium ion
a OH- =activities of hydroxide ion
BUFFER
These buffer species, like H+ and OH-, participates in formation of break
down of activated complexes of various reaction and determine their reaction
rate. These catalytic species are referred to as general acid-base catalysts. Studies
92
with phosphate buffer indicates that it enhance the degradation of various drug
substances such as carbenicillin etc.
pH
pH has a significant influence on the rate of decomposition of drugs that are
hydrolysed in solution and it usual to minimise this effect by formulating at the
pH of maximum stability using buffers.
The rate of reaction is, however, influenced not only by the catalytic effect
of hydrogen and hydroxyl ions (specific acid–base catalysis), but also by the
components of the buffer system (general acid–base catalysis).
The effect of the buffer components can be large.
For example, the hydrolysis rate of codeine in 0.05 M phosphate buffer at
pH 7 is almost 20 times faster than in unbuffered solution at this pH.
Complex pH rate profiles are seen when the ionisation of the drug changes
over the pH of measurement because of the differing susceptibility of the
unionised and ionised forms of the drug to hydrolysis.
We should also note that the oxidative degradation of some drugs in
solution may be pH dependent; for example, the oxidation of prednisolone is
base-catalysed.
Similarly, the oxidation of morphine occurs more rapidly in alkaline or
neutral solution than in acid solution. The reason for this may be the effect of pH
on the oxidation–reduction potential, E0, of the drug.
The photodegradation of several drugs is also pH-dependent.
For example, the photochemical decomposition of the benzodiazepine
derivative midazolam increases with pH, and
Ciprofloxacin is most sensitive to photodegradation at slightly basic pH
where the drug is in zwitterionic form, the stability increasing when the pH is
lowered to 3 – 4.
Temperature
Increase in temperature usually causes a very pronounced increase in the
hydrolysis rate of drugs in solution, a fact which is used to good effect in the
experimental studies of drug stability.

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 Such studies are usually carried out at high temperatures, say 60 or 80°C,
because the hydrolysis rate is greater at these temperatures and can therefore be
measured more easily.
Of course, if a formulation has to be heat sterilized then its stability will, in
any case, have to be measured at elevated temperatures.
The equation which describes the effect of temperature on decomposition,
and which shows us how to calculate the rate of breakdown at room temperature
from measurements at much higher temperatures, is the Arrhenius equation.
log k = log A – Ea/(2.303RT)
where Ea is the activation energy,
A is the frequency factor,
R is the gas constant (8.314 J mol–1 K–1) and
T is the temperature in kelvins

If a drug formulation is particularly unstable at room temperature, for

example, injections of penicillin, insulin, oxytocin and vasopressin, it should be
labelled with instructions to store in a cool place.
Ionic strength
We often need to add electrolytes to drug solutions, for example to control
their tonicity.
Consequently we must pay particular attention to any effect they may have
on stability.
The equation which describes the influence of electrolyte on the rate
constant is the Brønsted–Bjerrum equation:
Brønsted–Bjerrum equation:
log k = log k0 + 2AzAzB√μ
where,
zA and zB are the charge numbers of the two interacting ions,
A is a constant for a given solvent and temperature and

94
 μ is the ionic strength of the solution
Solvent effects
Since we are considering the hydrolysis of drugs it might seem that an
obvious way to reduce the breakdown would be to replace some or all of the
water in the system with a solvent such as alcohol or propylene glycol
Oxygen
The susceptibility of a drug to the presence of oxygen can be tested by
comparing its stability in ampoules purged with oxygen to that when it is stored
under nitrogen.
Drugs which have a higher rate of decomposition when exposed to oxygen
can be stabilised by replacing the oxygen in the storage container with nitrogen
or carbon dioxide.
These drugs should also be kept out of contact with heavy metals and
should be stabilised with antioxidants.
UNIT-III
IMPURITY PROFILING AND DEGRADENT
CHARACTERISATION:
Analytical Method Development and Validation
Overview
The primary focus of this chapter is on general approaches and
considerations toward development of chromatographic methods for separation,
identification, and quantification of pharmaceutical compounds, which may be
applied within the various functions in the drug development continuum.
The chapter also discusses the issues and parameters that must be considered in
the validation of analytical methods. At the end of the chapter, a scope of the
present research study is covered.

2.1 GENERAL INTRODUCTION

The number of drugs introduced into the market is increasing every year.
These drugs may be either new entities or partial structural modification of the
existing one. Very often there is a time lag from the date of introduction of
a drug into the market to the date of its inclusion in pharmacopoeias. This
happens because of the possible uncertainties in the continuous and wider usage
95
of these drugs, reports of new toxicities (resulting in their withdrawal from the
market), development of patient resistance and introduction of better drugs by
competitors. Under these conditions, standards and analytical procedures for
these drugs may not be available in the pharmacopoeias. There is a scope,
therefore to develop newer analytical methods for such drugs.
Analytical methods development and validation play important roles in the
discovery, development, and manufacture of pharmaceuticals. Pharmaceutical
products formulated with more than one drug, typically referred to as
combination products, are intended to meet previously unmet patients need by
combining the therapeutic effects of two or more drugs in one product. These
combination products can present daunting challenges to the analytical chemist
responsible for the development and validation of analytical methods. The
official test methods that result from these processes are used by quality control
laboratories to ensure the identity, purity, potency, and performance of drug
products.
Identification and quantification of impurities is a crucial task in
pharmaceutical process development for quality and safety. Related
components are the impurities in pharmaceuticals which are unwanted
chemicals that remain with the active pharmaceutical ingredients (APIs), or
develop during stability testing, or develop during formulation or upon aging of
both API and formulated APIs to medicines. The presence of these unwanted
chemicals even in small amounts may influence the efficacy and safety of the
pharmaceutical products. Various analytical methodologies are employed for the
determination of related components in pharmaceuticals. There is a great need
for development of new analytical methods for quality evaluation of new
emerging drugs.

Basic criteria for new method development of drug analysis:

 The drug or drug combination may not be official in any pharmacopoeias,

 A proper analytical procedure for the drug may not be available in the
literature due to patent regulations,
 Analytical methods may not be available for the drug in the form of a
formulation due to the interference caused by the formulation excipients,
 Analytical methods for the quantitation of the drug in biological fluids may
not be available,
 Analytical methods for a drug in combination with other drugs may not be
available,
 The existing analytical procedures may require expensive reagents and
solvents. It may also involve cumbersome extraction and separation
96
 procedures and these may not be reliable.
Method validation
The need to validate an analytical or bioanalytical method is
encountered by analysis in the pharmaceutical industry on an almost daily
basis, because adequately validated methods are a necessity for approvable
regulatory filings. What constitutes a validated method, however, is subject
to analyst interpretation because there is no universally accepted industry
practice for assay validation.

Literature survey
When develop an HPLC/UPLC method, the first step is always to consult
the chromatographic literature to find out if anyone else has done the analysis,
and how they did it. This will at least give an idea of the conditions that are
needed, and may save one having to do a great deal of experimental work.

2.2 PHARMACEUTICAL IMPURITIES

An impurity in a drug substance as defined by the International
Conference on Harmonisation (ICH) Guidelines is any component of the drug
substance that is not the chemical entity defined as the drug substance.
Similarly, an impurity in a drug product is any component of the drug
product that is not the chemical entity defined as the drug substance or an
excipient in the drug product. The safety of a drug product is dependent not
only on the toxicological properties of the active drug substance itself, but also
on the impurities that it contains. Therefore, identification, quantification, and
control of impurities in the drug substance and drug product, are an important
part of drug development and regulatory assessment. ICH Q3A and Q3B address
issues relevant to the regulation of impurities in the drug substance and drug
product. While many of the concepts and principles outlined in these documents
are applicable to Abbreviated New Drug Applications (ANDAs), certain
additional or modified restraints need to be considered. When FDA receives an
ANDA, a monograph defining certain key attributes of the drug substance and
drug product is frequently available in the United States Pharmacopeia (USP).
Sometimes, literature information on drug product impurities may also be
available. These public standards and literature data play a significant role in the
regulatory assessment process of an ANDA.

2.2.1 Classification of impurities

The safety and quality of the drug substance and drug product in a generic
product can be impacted by the presence of impurities. The nature and the
quantity of these impurities is governed by a number of factors, including
97
synthetic route of the drug substance, reaction conditions, quality of the starting
material of the drug substance, reagents, solvents, purification steps, excipients,
drug product manufacturing processes, packaging, and storage of the end
product. Based on ICH Q3A [1], drug substance impurities can be classified into
the following categories:
• Organic impurities (process- and drug-related)
• Inorganic impurities
• Residual solvents
Organic impurities can arise during the manufacturing process and/or
storage of the drug substance. They can be identified or unidentified, volatile or
non-volatile, and include:
• Starting materials
• By-products
• Intermediates
• Degradation products
• Reagents, ligands, and catalysts
Inorganic impurities can result from the manufacturing process. They are
normally known and identified and include:
• Reagents, ligands and catalysts
• Heavy metals or other residual metals
• Inorganic salts
• Other materials (e.g., filter aids, charcoal)
Solvents are inorganic or organic liquids used as vehicles for the
preparation of solutions or suspensions in the synthesis of the drug substance or
the manufacture of the drug product. Since these are generally of known toxicity,
the selection of appropriate limits for these solvents is easily accomplished (ICH
Q3C [3] on residual solvents).
2. Control of impurities
A specification is defined as a list of tests, references to analytical
procedures, and appropriate acceptance criteria that are numerical limits, ranges,
or other criteria for the tests described. It establishes the set of criteria to which a
drug substance or drug product should conform to be considered acceptable for
its intended use. “Conformance to specifications” means that the drug substance
and/or drug product, when tested according to the listed analytical procedures,
will meet the listed acceptance criteria. Specifications are critical quality
standards that are proposed and justified by the manufacturer and approved by
regulatory authorities as conditions of approval.

2.2.3 Listing of impurities in drug substance specification

98
 The specifications for a drug substance include a list of impurities.
Stability studies, chemical development studies, routine batch analyses, and
scientific appraisal of potential by- products from synthetic steps and degradation
pathways, can be used to predict those impurities likely to occur in the drug
substance. The drug substance specification includes, where applicable, a list of
the following types of impurities:
 Organic impurities
 Each identified specified impurity
 Each specified unidentified impurity
 Total impurities
 Residual solvents
 Inorganic impurities
2.2.4 Listing of impurities in drug product specification
The specification for a drug product should include a list of degradation
products. Stability studies, chemical development studies, and routine batch
analyses can be used to predict the degradation profile for the commercial
product. The drug product specification includes, where applicable, types of
degradation products are:
• Each specified identified degradation product
• Each specified unidentified degradation product
• Total degradation products

2.3 ROLE OF DEGRADANT PROFILING IN ACTIVE

PHARMACEUTICAL INGREDIENTS AND DRUG PRODUCTS
2.3.1 Regulatory Requirements
From a regulatory perspective, forced degradation studies provide data to
support the followings:
• Identification of possible degradants
• Degradation pathways and intrinsic stability of the drug molecule
• Validation of stability indicating analytical procedures.

Issues addressed in regulatory guidances include:

• Forced degradation studies are typically carried out using one batch of
material.
• Forced degradation conditions are more severe than accelerated stability
testing such as 50°C; ≥75% relative humidity; in excess of ICH light
conditions; high and low pH, oxidation, etc.
• Photostability should be an integral part of forced degradation study design.
• Degradation products that do not form in accelerated or long term stability
99
may not have to be isolated or have their structure determined.
• Mass balance should be considered.

Issues not specifically addressed in regulatory guidance:

•Exact experimental conditions for forced degradation studies (temperatures,
duration, extent of degradation, etc.) are not specified.
•Experimental design is left to the applicant's discretion. There is guidance
available from the FDA as well as from private industry on regulatory
requirements for IND and NDA filings [18].
2.3.2 Forced degradation timing and strategy
The requirements for forced degradation testing depend on project needs
and the stage of development of the compound. For example, pre-clinical
through phase-II project needs dictate intense method development, and the rate
of compound attrition is high. Therefore, when developing a rational study
design, forced degradation deliverables should be focused on method
development activities, and not isolation and identification of degradants.
The focus of stress testing should be directed to characterization and
elucidation of degradants

Impurity profile: Significance in Active Pharmaceutical Ingredient

1. Introduction
There is an ever increasing interest in impurities present in API’s. Recently,
not only purity profile but also impurity profile has become essential as per
various regulatory requirements. In the pharmaceutical world, an impurity is
considered as any other organic material, besides the drug substance, or
ingredients, arise out of synthesis or unwanted chemicals that remains with
API’s. The impurity may be developed either during formulation, or upon
aging of both API’s and formulated API’s in medicines. A good illustration of
this definition may be identification of impurity in API’s like 1-(1, 2, 3, 5, 6, 7-
hexahydro-s-indacen-4-yl)-3-4[-1-hydroxy-1-methyl-ethyl)-furan-2-
sulphonylurea using Multidisciplinary approach [1]. The presence of these
unwanted chemicals, even in small amount, may influence the efficacy and
safety of the pharmaceutical products. Impurity profiling (i.e., the identity as well
as the quantity of impurity in the pharmaceuticals), is now gaining critical
attention from regulatory authorities. The different Pharmacopoeias, such as the
British Pharmacopoeia (BP), United States Pharmacopeia (USP), and Indian
Pharmacopoeia (IP) are slowly incorporating limits to allowable levels of
impurities present in the API’s or formulations.

The International Conference on Harmonization of Technical Requirements

100
for Registration of Pharmaceuticals for Human Use (ICH) has also
published guidelines for validation of methods for analysing impurities in
new drug substances, products, residual solvents and microbiological
impurities [2-5].

A number of articles [8-10] have stated guidelines and designed approaches

for isolation and identification of process-related impurities and degradation
products, using Mass spectrometry (MS), Nuclear Magnetic Resonance (NMR),
High Performance Liquid Chromatography (HPLC), Fourier Transform Ion
Cyclotron Resonance Mass Spectrometry (FTICR-MS), and Tandem Mass
Spectrometry for pharmaceutical substances.

Present article reveals different impurities found in the API’s, methods for
identifying them and the possible measures to deal with the interferences caused
by them.

Impurity profile is the description of identified and unidentified impurities

present in new drug substances. Impurities can be described as shown in Table 1.

Impurities have been named differently or classified as per the ICH as

follows;
a) Common names
- By-products
- Degradation products
- Penultimate intermediates
- Related products
- Transformation products

Sources of Impurities
From the preceeding discussion, it is clear that impurities can originate
from several sources; such as; a) Crystallization-related impurities,
b)Stereochemistry-related impurities, c) Residual solvents, d) Synthetic
intermediates and by-products, e) Formulation-related impurities, g) Impurities
arising during storage, h) Method related impurity, I) Mutual interaction amongst
ingredients, h) Functional group-related typical degradation.

Analytical method development

New drug development requires meaningful and reliable analytical data to
be produced at various stages of the development.
a) Sample set selection for analytical method development
101
b) Screening of Chromatographic conditions and Phases, typically using the
linear- solvent-strength model of gradient elution
c) Optimization of the method to fine-tune parameters related to ruggedness and
robustness
The impurities can be identified predominantaly by following methods;
• Reference standard method
• Spectroscopic method
• Separation method
• Isolation method
• Characterization method

5. Reference standard method

The key objective of this is to provide clarity to the overall life cycle,
qualification and governance of reference standards used in development and
control of new drugs. Reference standards serve as the basis of evaluation of
both process and product performance and are the benchmarks for assessment of
drug safety for patient consumption. These standards are needed, not only for the
active ingredients in dosage forms but also for impurities, degradation products,
starting materials, process intermediates, and excipients.

6. Spectroscopic methods
The UV, IR, MS, NMR and Raman spectroscopic methods are routinely
being used for characterizing impurities.

7. Separation methods
The Capillary electrophoresis (CE), Chiral Separations, Gas
Chromatography (GC), Supercritical Fluid Chromatography (SFC), TLC,
HPTLC, HPLC are regularly being used for separation of impurities and
degradation products

8.Isolation methods
It is often necessary to isolate impurities. But if the instrumental
methods are used, isolation of impurities is avoided as it directly characterizes
the impurities.
Generally, chromatographic and non-chromatographic techniques are used
for isolation of impurities prior its characterization. The term ‘chromatographic
reactor’ refers to the use of an analytical-scale column as both a flow-through
reactor, and simultaneously, as separation medium for the reactant(s) and
product(s). By using an HPLC, chromatographic reactor approach, the
102
solution-phase hydrolysis kinetics of the Aprepitant (EmendTM) prodrug,
fosaprepitant dimeglumine, were investigated. In loratidine, impurity found was
ofloratidine, other examples include celecoxib, and amikacin.
A list of methods that can be used for isolation of impurities is given
below.
• Solid-phase extraction methods
• Liquid-liquid extraction methods
• Accelerated solvent extraction methods
• Supercritical fluid extraction
• Column chromatography
• Flash chromatography
• TLC
• GC
• HPLC
• HPTLC
• Capillary electrophoresis (CE)
• Supercritical fluid chromatography (SFC)
Applications
Numerous applications have been sought in the areas of drug designing and
in monitoring quality, stability, and safety of pharmaceutical compounds,
whether produced synthetically, extracted from natural products or produced by
recombinant methods. The applications include alkaloids, amines, amino acids,
analgesics, antibacterials, anticonvulsants, antidepressant, tranquilizers,
antineoplastic agents, local anesthetics, macromolecules, steroids,
miscellaneous .
Shelf Life Estimation of Pharmaceutical Products
Learn how the shelf life of the pharmaceutical products and substances is
determined according the ICH guidelines. All pharmaceutical drugs degrade with
the time forming the byproducts. These byproducts may harmful for health of
the patients consuming the Shelf life of the pharmaceutical products is the time
period for which the product maintains its identity and quality when stored at the
conditions defin the label of the product.
It is important to set the time frame to consume the pharmaceutical
products. ICH Q1E guideline provides guidance for the estimation of the shelf
life of the pharmaceutical products and substances. Shelf life is determined after
the evaluation of whole stability data of the product.
Related: Mean Kinetic Temperature (MKT) in Stability Studies
Minimum data of three batches is used to estimate the shelf life of
pharmaceutical products. For the products stored at room temperature the
assessment should be started from the occurrence of the significant change in
103
product stored in accelerated conditions. If no significant change is found in the
accelerated conditions then shelf life would be depend upon the long term
storage conditions data.
If long term and accelerated conditions data shows very little change or no
change at all then it might be assumed that the product would be stable during the
proposed shelf life period. In this case there is no requirement of the statistical
analysis and the justification for the same should be given. In this case the shelf
life of the product can be proposed twice but should not be more than 12 months
of the period covered by the long term evaluation data.
Related: Impurity Profiling of Drug Substances in Pharmaceuticals
If long term condition or accelerated condition data shows change then
statistical analysis is used to estimate the shelf-life of the drug product. Whe
statistical calculations cannot be done by the data, the shelf life period can be
defined 1.5 time of the period covered by the long term data.
When the data is sufficient for the statistical calculations shelf-life would
be defined double but not more than 12 months from the period covered b long
term data. Details can be read in ICH Q1E guidelines.

WHO AND ICH STABILITY TESTING GUIDELINES,STABILITY

ZONES,STEPS IN DEVELOPMENT,PRACTICAL CONSIDERATION
Why Stability?
To Provide a evidence on how the quality of a drug substance or drug product
varies with time under the influence of a variety of environmental factors such as
temperature, humidity, and light. Establish a
• re-test period for the drug substance or a
• shelf life for the drug product and
• recommended storage conditions
Because physical, chemical or microbiological changes might impact the
• efficiency and security of the final product
Where and Why?
Stability Studies are preformed on
 Drug Substances (DS) the unformulated drug substance that may
subsequently be formulated with excipients to produce the dosage form
 Drug Products (DP) the dosage form in the final immediate packaging
intended for marketing

What are changes?

104
 CHEMICAL BIOLOGICAL
PHYSICAL
Appearance Increase in degradation Growth of micro
products organism
Melting point Decrease of assay
Clarity & odour
Crystal
modification
Particle size
Water
Stability studies at different stages
1. Stress- and accelerated Testing with drug substances
2. Stability on pre-formulation batches
3. Stress testing on scale-up Batches
4. Accelerated and long term testing for registration
5. On-going Stability testing
6. Follow-up Stabilities

WHO GUIDELINES FOR STABILITY STUDIES:

 These guidelines seek to exemplify the core stability data package required
for registration of active pharmaceutical ingredients (APIs) and finished
pharmaceutical products (FPPs)
 These guidelines apply to new and existing APIs and address information
to be submitted in original and subsequent applications for marketing
authorization of their related FPP for human use. These guidelines are not
applicable to stability testing for biologicals.
 The purpose of stability testing is to provide evidence of how the quality of
an API or FPP varies with time under the influence of a variety of
environmental factors such as temperature, humidity and light.
 The stability programme also includes the study of product-related factors
that influence its quality, for example, interaction of API with excipients,
container closure systems and packaging materials.
 In fixed-dose combination FPPs (FDCs) the interaction between two or
more APIs also has to be considered. As a result of stability testing a re-test
period for the API (in exceptionalcases, e.g. for unstable APIs, a shelf-life
is given) or a shelf-life for the FPP can be established and storage
conditions can be recommended.

105
 STABILITY GUIDELINES FOR ACTIVE PHARMACEUTICAL
INGREDIENT AND FINISHED PHARMACEUTICAL PRODUCT

Active pharmaceutical Finished pharmaceutical

ingredient ingredient
Stress testing Stress testing
Selection of batches Selection of batches
Container closure system Container closure system
Specification Specification
Testing frequency Testing frequency
Storage Condition Storage Condition
Evaluation Evaluation
Statements &labelling Statements &labelling
Ongoing stability studies Ongoing stability studies
Inuse stability
variations

STRESS TESTING:
Stress testing of the API can help identify the likely degradation products,
which, in turn, can help establish the degradation pathways and the intrinsic
stability of the molecule and validate the stability-indicating power of the
analytical procedures used.
The nature of the stress testing will depend on the individual API and the
type of FPP involved. Stress testing may be carried out on a single batch of the
API. It should include the effect of temperature (in 10 °C increments (e.g. 50
°C, 60 °C,etc.) above the temperature used for accelerated testing), humidity
(e.g. 75% relative humidity (RH) or greater) and, where appropriate, oxidation
and photolysis on the API. The testing should also evaluate the susceptibility of
the API to hydrolysis across a justifi ed range of pH values when in solution or
suspension.
SELECTION OF BATCHES:
Data from stability studies on at least three primary batches of the API
should normally be provided.
The batches should be manufactured to a minimum of pilot scale by the
same synthesis route as production batches, and using a method of manufacture
and procedure that simulates the final process to be used for production batches.
The overall quality of the batches of API placed on stability studies should be
representative of the quality of the material to be made on a production scale.
For existing active substances that are known to be stable, data from at least
two primary batches should be provided.
106
CONTAINER CLOSURE SYSTEM
The stability studies should be conducted on the API packaged in a
container closure system that is the same as, or simulates, the packaging
proposed for storage and distribution.

SPECIFICATION
 Stability studies should include testing of those attributes of the API that
are susceptible to change during storage and are likely to influence quality,
safety and/or efficacy.
 The testing should cover, as appropriate, the physical, chemical, biological
and microbiological attributes. Validated stability indicating analytical
procedures should be applied. Whether and to what extent replication
should be performed will depend on the results from validation studies .

TESTING FREQUENCY
 For long-term studies, frequency of testing should be suffi cient to establish
the stability profile of the API. For APIs with a proposed re-test period or
shelf-life of at least 12 months, the frequency of testing at the long-term
storage condition should normally be every three months over the fi rst
year, every six months over the second year, and annually thereafter
throughout the proposed re-test period or shelf-life.
 At the accelerated storage condition, a minimum of three time points,
including the initial and fi nal time points (e.g. 0, 3 and 6 months), from a
sixmonth study is recommended.
 Where it is expected (based on development experience) that results from
accelerated studies are likely to approach significant change criteria,
increased testing should be conducted either by adding samples at the final
time point or by including a fourth time point in the study design.
 When testing at the intermediate storage condition is called for as a result
of signifi cant change at the accelerated storage condition, a minimum of
four time points, including the initial and final time points (e.g.0, 6, 9 and
12 months), from a 12-month study is recommended.

STORAGE CONDITION:
 In general an API should be evaluated under storage conditions (with
appropriate tolerances) that test its thermal stability and, if applicable, its
sensitivity to moisture.

107
  The storage conditions and the lengths of studies chosen should be
sufficient to cover storage and shipment. Storage condition tolerances are
defined as the acceptable variations in temperature and relative humidity of
storage facilities for stability studies.
General case:
STUDY STORAGE MINIMUM TIME
CONDITION PERIOD COVERED
BY DATA AT
SUBMISSION
Long term 25c±2c/60%RH±5%RH 12 Months or 6
or months
30c±2c/65%RH±5%RH
or
30c±2c/75%RH±5%RH

Intermediate 30c±2c/65%RH±5%RH 6Months

Accelerated 40c±2c/75%RH±5%RH 6Months

Active pharmaceutical ingredients intended for storage in a refrigerator:

Study Storage condition Minimum time
period covered by
data at submission
Long term 5c±3c 12 months
Accelerated 25c±2c/60%RH±5%RH
or
30c±2c/65%RH±5%RH 6 months
or
30c±2c/75%RH±5%RH

Active pharmaceutical ingredients intended for storage in a freezer:

Study Storage condition Minimum time period
covered by data at
submission
Long term -20c±5c 12 months

Evaluation:
 The purpose of the stability study is to establish, based on testing a
minimum of the number of batches unless otherwise justified and
108
 authorized, of the API and evaluating the stability information (including,
as appropriate, results of the physical, chemical, biological and
microbiological tests), a re-test period applicable to all future batches of the
API manufactured under similar circumstances.
 The degree of variability of individual batches affects the confidence that a
future production batch will remain within specification throughout the
assigned re-test period.
 An approach for analysing the data on a quantitative attribute that is
expected to change with time is to determine the time at which the 95%
one-sided confidence limit for the mean curve intersects the acceptance
criterion.
 If analysis shows that the batch-to-batch variability is small, it is
advantageous to combine the data into one overall estimate.
 This can be done by first applying appropriate statistical tests (e.g. p values
for level of significance of rejection of more than 0.25) to the slopes of the
regression lines and zero time intercepts for the individual batches.
 If it is inappropriate to combine data from several batches, the overall re-
test period should be based on the minimum time a batch can be expected
to remain within acceptance criteria.
 The nature of any degradation relationship will determine whether the data
should be transformed for linear regression analysis.
 Usually the relationship can be represented by a linear, quadratic or cubic
function on an arithmetic or logarithmic scale.
 As far as possible, the choice of model should be justified by a physical
and/or chemical rationale and should also take into account the amount of
available data (parsimony principle to ensure a robust prediction).
 Statistical methods should be employed to test the goodness of fit of the
data on all batches and combined batches (where appropriate) to the
assumed degradation line or curve.
 Limited extrapolation of the long-term data from the long-term storage
condition beyond the observed range to extend the re-test period can be
undertaken if justified.
 This justification should be based on what is known about the mechanism
of degradation, the results of testing under accelerated conditions, the
goodness of fi t of any mathematical model, batch size and existence of
supporting stability data.
 However, this extrapolation assumes that the same degradation relationship
will continue to apply beyond the
observed data.
109
  Any evaluation should cover not only the assay but also the levels of
degradation products and other appropriate attributes.
Where appropriate, attention should be paid to reviewing the adequacy of
evaluation linked to FPP stability and degradation “behaviour” during the testing.
Statements and labelling:
A storage statement should be established for display on the label based on
the stability evaluation of the API. Where applicable specific instructions should
be provided, particularly for APIs that cannot tolerate freezing or excursions in
temperature. Terms such as “ambient conditions” or “room temperature” should
be avoided. A re-test period should be derived from the stability information, and
a retest date should be displayed on the container label if appropriate.
Stability Guidelines for Finished Pharmaceutical Ingredient:
General:
The design of the stability studies for the FPP should be based on
knowledge of the behaviour and properties of the API, information from stability
studies on the API and on experience gained from preformulation studies and
investigational FPPs.
Selection of batches:
 Data from stability studies should be provided on at least three primary
batches of the FPP.
 The primary batches should be of the same formulation and packaged in the
same container closure system as proposed for marketing.
 The manufacturing process used for primary batches should simulate that
be applied to production batches and should provide product of the same
and meeting the same specifi cation as that intended for marketing.
 In the case of conventional dosage forms with APIs that are known to be
stable, data from at least two primary batches should be provided. Two of
the three batches should be at least pilot-scale batches and the third one can
be smaller, if justified.
 Where possible, batches of the FPP should be manufactured using different
batches of the API(s).
 Stability studies should be performed on each individual strength, dosage
form and container type and size of the FPP unless bracketing or matrixing
is applied.
Container closure system:
 Stability testing should be conducted on the dosage form packaged in the
container closure system proposed for marketing.
 Any available studies carried out on the FPP outside its immediate
container or in other packagin materials can form a useful part of the stress
110
 testing of the dosage form or can be considered as supporting information,
respectively.
Storage condition:
 In general an FPP should be evaluated under storage conditions with
specified tolerances that test its thermal stability and, if applicable, its
sensitivity to moisture or potential for solvent loss.
 The storage conditions and the lengths of studies chosen should be
sufficient to cover storage, shipment and subsequent use with due regard to
the climatic conditions in which the product is intended to be marketed.
 Photostability testing, which is an integral part of stress testing, should be
conducted on at least one primary batch of the FPP if appropriate.
General Case:
Study Storage Condition Minimum time period
covered by data at
submission
Long term 25c±2c/60%RH±5%RH 12 Months or 6
or months
30c±2c/65%RH±5%RH
or
30c±2c/75%RH±5%RH

Intermediate 30c±2c/65%RH±5%RH 6Months

Accelerated 40c±2c/75%RH±5%RH 6Months

FPP’s Stored in Semipermeable containers:

 Aqueous-based products packaged in semi-permeable containers should be
evaluated for potential water loss in addition to physical, chemical,
biological and microbiological stability.
 This evaluation can be carried out under conditions of low relative
humidity, as discussed below.
 Ultimately it should be demonstrated that aqueous-based FPPs stored in
semi-permeable containers could withstand environments with low relative
humidity.
 Other comparable approaches can be developed and reported for non
aqueous, solvent-based products.

STUDY STORAGE MINIMUM TIME

CONDITION PERIOD COVERED
BY DATA AT
111
 SUBMISSION
Long term 25c±2c/60%RH±5%RH 12 Months
or
30c±2c/65%RH±5%RH

Intermediate 30c±2c/65%RH±5%RH 6Months

Accelerated 40c±2c/NMT 25%RH 6Months

FPP’s Intended for stored in a refrigerator:

Study Storage condition Minimum time

period covered by
data at submission
Long term 5c±3c 12 months
Accelerated 25c±2c/60%RH±5%RH
or
30c±2c/65%RH±5%RH 6 months
or
30c±2c/75%RH±5%RH

FPP’s Intended for stored in a Freezer:

Study Storage condition Minimum time period

covered by data at
submission
Long term -20c±5c 12 months

Evaluation:
 A systematic approach should be adopted to the presentation and evaluation
of the stability information, which should include, as appropriate, results
from the physical, chemical, biological and microbiological tests, including
particular attributes of the dosage form.
 The purpose of the stability study is to establish, based on testing a
minimum number of batches of the FPP . The degree of variability of
individual batches affects the confidence that a future production batch will
remain within specifi cation throughout its shelf-life.

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  Where the data show so little degradation and so little variability that it is
apparent from looking at the data that the requested shelf-life will be
granted, it is normally unnecessary to go through the statistical analysis.
 An approach for analysing the data on a quantitative attribute that is
expected to change with time is to determine the time at which the 95%
one-sided confidence limit for the mean curve intersects the acceptance
criterion. If analysis shows that the batch-to-batch variability is small, it is
advantageous to combine the data into one overall estimate. This can be
done by first applying appropriate statistical tests (e.g. p values for level of
significance of rejection of more than 0.25) to the slopes of the regression
lines and zero time intercepts for the individual batches.
 If it is inappropriate to combine data from several batches, the overall shelf-
life should be based on the minimum time a batch can be expected to
remain within acceptance criteria.
 The nature of any degradation relationship will determine whether the data
should be transformed for linear regression analysis. Usually the
relationship can be represented by a linear, quadratic or cubic function on
an arithmetic or logarithmic scale.
 As far as possible, the choice of model should be justified by a physical
and/or chemical rationale and should also take into account the amount of
available data (parsimony principle to ensure a robust prediction).
 Statistical methods should be employed to test the goodness of fit of the
data on all batches and combined batches (where appropriate) to the
assumed degradation line or curve.
 Limited extrapolation of the long-term data from the long-term storage
condition beyond the observed range to extend the shelf-life can be
undertaken, if justified.
 This justification should be based on what is known about the mechanism
of degradation, the results of testing under accelerated conditions, the
goodness of fi t of any mathematical model, batch size and the
existence of supporting stability data. However, this extrapolation assumes
that the same degradation relationship will continue to apply beyond the
observed data.
 Any evaluation should consider not only the assay but also the degradation
products and other appropriate attributes. Where appropriate, attention
should be paid to reviewing the adequacy of evaluation linked to FPP
stability and degradation “behaviour” during the testing

What is ICH GuideLines?

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 ICH stands for International Council for Harmonization of technical
requirements for registration of pharmaceuticals for human use. ICH is a joint
initiative involving both regulators and industry as equal partners in the scientific
and technical discussions of the testing procedures that are required to ensure and
asses the safety, quality and efficacy of medicines.

Drug stability:
Stability of pharmaceutical product may be defined as the capability of a
particular formulation in a specific container/closer system to remain within its
physical, chemical, microbiological therapeutic and toxicological specification.

Q1A-Q1F STABILITY

• Q1A(R2)Stability Testing of New Drug Substances and Products

• Q1B Stability Testing : Photostability Testing of New Drug Substances and
Products
• Q1C Stability Testing for New Dosage Forms
• Q1D Bracketing and Matrixing Designs for Stability Testing of New Drug
Substances and Products
• Q1E Evaluation of Stability Data
• Q1F Stability Data Package for Registration Applications in Climatic
Zones III and IV
• (Q2) Validation of Analytical procedures
• (Q3) Impurities
• (Q5) Biotechnological products
• (Q6) Specifications

STABILITY TEST PARAMETERS FOR VARIOUS TYPES OF

PRODUCTS
 Tablets – Appearance, colour, odour, assay, disintegration/dissolution,
moisture and friability or hardness testing.
 Hard gelatin capsules - Appearance, colour, odour of contents, assay,
disintegration/dissolution, moisture and microbial limits
 Soft gelatin capsules - Appearance, colour, odour of contents, assay,
disintegration/dissolution, moisture, microbial limits, ph , leakage and
pellicle formation.
 Emulsions – Appearance including phase separation, colour, odour, assay,
pH, viscosity, preservative content, weight loss and microbial limits.
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  Suppositories – Appearance, colour, assay, particle size, softening range,
appearance, dissolution and microbial limits.
 Small volume parenteral- Drug injection – Appearance, colour, assay, ph,
preservative, content, particulate matter, sterility and pyrogenicity.
 Large volume parenteral - Appearance, colour, assay, ph, preservative
content, particulate matter, sterility and pyrogenicity
 Transdermals – Appearance, assay, leakage, microbial limit/sterility, peel
and adhesive forces, drug release rate.

Stability zones
Climatic Zone Definition Storage Condition
1 Temperate climate 21c/45%RH
2 Subtropical 25C/60%RH
3 Hot and Dry Climate 30C/35%RH
4A Hot and Humidity 30C/65%RH
Climate
4B Hot and Very 30C/75%RH
Humidity climate

Q1A(R2)Stability Testing of New Drug Substances and Products

Objective of the guideline:
The following guideline is a revised version of the ICH Q1A guideline and
defines the stability data package for a new drug substance or drug product that is
sufficient for a registration application within the three regions of the EC, Japan,
and the United States. It does not seek necessarily to cover the testing for
registration in or export to other areas of the world.
The guideline seeks to exemplify the core stability data package for new
drug substances and products, but leaves sufficient flexibility to encompass the
variety of different practical situations that may be encountered due to specific
scientific considerations and characteristics of the materials being evaluated.
Alternative approaches can be used when there are scientifically justifiable
reasons.
Scope of the Guideline
The guideline addresses the information to be submitted in registration
applications for new molecular entities and associated drug products. This
guideline does not currently seek to cover the information to be submitted for
abbreviated or abridged applications, variations, clinical trial applications, etc.
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 Specific details of the sampling and testing for particular dosage forms in
their proposed container closures are not covered in this guideline.
Further guidance on new dosage forms and on biotechnological/biological
products can be found in ICH guidelines Q1C and Q5C, respectively.
General Principles
The purpose of stability testing is to provide evidence on how the quality of
a drug substance or drug product varies with time under the influence of a variety
of environmental factors such as temperature, humidity, and light, and to
establish a re-test period for the drug substance or a shelf life for the drug product
and recommended storage conditions.
The choice of test conditions defined in this guideline is based on an
analysis of the effects of climatic conditions in the three regions of the EC, Japan
and the United States. The mean kinetic temperature in any part of the world can
be derived from climatic data, and the world can be divided into four climatic
zones, I-IV. This guideline addresses climatic zones I and II. The principle has
been established that stability information generated in any one of the three
regions of the EC, Japan and the United States would be mutually acceptable to
the other two regions, provided the information is consistent with this guideline
and the labeling is in accord with national/regional requirements.

Q1C Stability Testing for New Dosage Forms:

A new dosage form is defined as a drug product which is a different
pharmaceutical product type, but contains the same active substance as included
in the existing drug product approved by the pertinent regulatory authority.
Such pharmaceutical product types include products of different
administration route (e.g., oral to parenteral), new specific functionality/delivery
systems (e.g., immediate release tablet to modified release tablet) and different
dosage forms of the same administration route (e.g., capsule to tablet, solution to
suspension).
Stability protocols for new dosage forms should follow the guidance in the
parent stability guideline in principle. However, a reduced stability database at
submission time (e.g., 6 months accelerated and 6 months long term data from
ongoing studies) may be acceptable in certain justified cases.
Q1D Bracketing and Matrixing Designs for Stability Testing of New Drug
Substances and Products

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 This guideline is intended to address recommendations on the application
of bracketing and matrixing to stability studies conducted in accordance with
principles outlined in the ICH Q1A(R) Harmonised Tripartite guideline on
Stability Testing of New Drug Substances and Products (hereafter referred to as
the parent guideline).
Bracketing is the design of a stability schedule such that only samples on
the extremes of certain design factors (e.g., strength, container size and/or fill)
are tested at all time points as in a full design. The design assumes that the
stability of any intermediate levels is represented by the stability of the extremes
tested.
The use of a bracketing design would not be considered appropriate if it
cannot be demonstrated that the strengths or container sizes and/or fills selected
for testing are indeed the extremes.
Matrixing is the design of a stability schedule such that a selected subset of
the total number of possible samples for all factor combinations would be tested
at a specified time point. At a subsequent time point, another subset of samples
for all factor combinations would be tested. The design assumes that the stability
of each subset of samples tested represents the stability of all samples at a given
time point. The differences in the samples for the same drug product should be
identified as, for example, covering different batches, different strengths,
different sizes of the same container closure system, and possibly, in some cases,
different container closure systems.
When a secondary packaging system contributes to the stability of the drug
product, matrixing can be performed across the packaging systems.
Each storage condition should be treated separately under its own matrixing
design. Matrixing should not be performed across test attributes. However,
alternative matrixing designs for different test attributes can be applied if
justified.
Q1E Evaluation of Stability Data
This guideline is intended to provide recommendations on how to use
stability data generated in accordance with the principles detailed in the ICH
guideline “Q1A(R) Stability Testing of New Drug Substances and Products”
(hereafter referred to as the parent guideline) to propose a retest period or shelf
life in a registration application. This guideline describes when and how
extrapolation can be considered when proposing a retest period for a drug
substance or a shelf life for a drug product that extends beyond the period

117
covered by “available data from the stability study under the long-term storage
condition” (hereafter referred to as long-term data).
The design and execution of formal stability studies should follow the
principles outlined in the parent guideline. The purpose of a stability study is to
establish, based on testing a minimum of three batches of the drug substance or
product, a retest period or shelf life and label storage instructions applicable to all
future batches manufactured and packaged under similar circumstances. The
degree of variability of individual batches affects the confidence that a future
production batch will remain within acceptance criteria throughout its retest
period or shelf life.

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 Although normal manufacturing and analytical variations are to be
expected, it is important that the drug product be formulated with the intent to
provide 100 percent of the labeled amount of the drug substance at the time of
batch release. If the assay values of the batches used to support the registration
application are higher than 100 percent of label claim at the time of batch release,
after taking into account manufacturing and analytical variations, the shelf life
proposed in the application can be overestimated. On the other hand, if the assay
value of a batch is lower than 100 percent of label claim at the time of batch
release, it might fall below the lower acceptance criterion before the end of the
proposed shelf life.
A systematic approach should be adopted in the presentation and evaluation
of the stability information. The stability information should include, as
appropriate, results from the physical, chemical, biological, and microbiological
tests, including those related to particular attributes of the dosage form (for
example, dissolution rate for solid oral dosage forms). The adequacy of the mass
balance should be assessed. Factors that can cause an apparent lack of mass
balance should be considered, including, for example, the mechanisms of
degradation and the stability-indicating capability and inherent variability of the
analytical procedures.
The basic concepts of stability data evaluation are the same for single-
versus multi-factor studies and for full- versus reduced-design studies. Data from
formal stability studies and, as appropriate, supporting data should be evaluated
to determine the critical quality attributes likely to influence the quality and
performance of the drug substance or product. Each attribute should be assessed
separately, and an overall assessment should be made of the findings for the
purpose of proposing a retest period or shelf life. The retest period or shelf life
proposed should not exceed that predicted for any single attribute.
The decision tree in Appendix A outlines a stepwise approach to stability data
evaluation and when and how much extrapolation can be considered for a
proposed retest period or shelf life. Appendix B provides (1) information on how
to analyze long-term data for appropriate quantitative test attributes from a study
with a multi-factor, full or reduced design, (2) information on how to use
regression analysis for retest period or shelf life estimation, and (3) examples of
statistical procedures to determine poolability of data from different batches or
other factors. Additional guidance can be found in the references listed; however,
the examples and references do not cover all applicable statistical approaches.
In general, certain quantitative chemical attributes (e.g., assay, degradation
products, preservative content) for a drug substance or product can be assumed to
follow zero-order kinetics during long-term storage. Data for these attributes are
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therefore amenable to the type of statistical analysis described in Appendix B,
including linear regression and poolability testing. Although the kinetics of other
quantitative attributes (e.g., pH, dissolution) is generally not known, the same
statistical analysis can be applied, if appropriate. Qualitative attributes and
microbiological attributes are not amenable to this kind of statistical analysis.
The recommendations on statistical approaches in this guideline are not
intended to imply that use of statistical evaluation is preferred when it can be
justified to be unnecessary. However, statistical analysis can be useful in
supporting the extrapolation of retest periods or shelf lives in certain situations
and can be called for to verify the proposed retest periods or shelf lives in other
cases.
Q1F Stability Data Package for Registration Applications in Climatic Zones
III and IV
ICH Q1 F Stability Data Package for Registration Applications in Climatic
Zones III and IV defined storage conditions for stability testing in countries
located in Climatic Zones III (hot and dry) and IV (hot and humid), i.e. countries
not located in the ICH regions and not covered by ICH Q1 A (R2) Stability
Testing for New Drug Substances and Drug Products.
ICH Q1 F described harmonised global stability testing requirements in
order to facilitate access to medicines by reducing the number of different
storage conditions.
In the course of the discussions which led to the development of the
guideline, WHO conducted a survey amongst their member states to find
consensus on 30°C/65% RH as the long-term storage conditions for hot and
humid regions. As no significant objections were raised in this survey, 30°C/65%
RH was defined as the long-term storage condition for Climatic Zone III/IV
countries in ICH Q1F.
The document was adopted by the ICH Steering Committee in February
2003 and subsequently implemented in the ICH regions.
However, based on new calculations and discussions, some countries in
Climatic Zone IV have expressed their wish to include a larger safety margin for
medicinal products to be marketed in their region than foreseen in ICH Q1F. As a
consequence, several countries and regions have revised their own stability
testing guidelines, defining up to 30°C/75 % RH as the long-term storage
conditions for hot and humid regions.
Due to this divergence in global stability testing requirements, the ICH
Steering Committee has decided to withdraw ICH Q1F and to leave definition of
storage conditions in Climatic Zones III and IV to the respective regions and
WHO.
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 In assessing the impact of the withdrawal of ICH Q1F on intermediate
testing conditions defined in ICH Q1A (R2), the decision was reached to retain
30°C/65%RH. However, regulatory authorities in the ICH regions have agreed
that the use of more stringent humidity conditions such as 30°C/75% RH will be
acceptable should the applicant decide to use them.
IMPURITY PROFILING
The description, characterization and quantitation of the identified and
unidentified impurities present in new drug substances is known as impurity
profile. A general scheme is set for the estimation of the impurity of bulk drug
substances by the rational use of chromatographic, spectroscopic and analytical
techniques. The impurity may be developed either during formulation, or upon
aging of both API’s and formulated API’s in medicines. The presence of these
unwanted chemicals, even in small amount, may influence the efficacy and safety
of the pharmaceutical products. Any material that affects the purity of the
material of interest viz. active ingredient or drug substance. The impurities are
not necessarily always inferior. From the standpoint of its usage, the drug
substance is compromised in terms of purity even if it contains another material
with superior pharmacological or toxicological properties.
INTRODUCTION:
Impurity is defined as any substance coexisting with the original drug, such
as starting material or intermediates or that is formed, due to any side reactions.
Impurity can be of three types: Impurities closely related to the product and
coming from the chemical or from the biosynthetic route itself, Impurities
formed due to spontaneous decomposition of the drug during the storage or on
exposure to extreme conditions, or the precursors which may be present in the
final product as impurities.
Impurities present in excess of 0.1% should be identified and quantified by
selective methods. The suggested structures of the impurities can be synthesized
and will provide the final evidence for their structures, previously determined by
spectroscopic methods. Therefore it is essential to know the structure of these
impurities in the bulk drug in order to alter the reaction condition and to reduce
the quantity of impurity to an acceptable level. Isolation, identification and
quantification of impurities help us in various ways, to obtain a pure substance
with less toxicity and, safety in drug therapy.
Quantitative determination of these impurities could be used as a method
for the quality control and validation of drug substances. Regulatory authorities
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such as US FDA, cGMP, TGA, and MCA insist on the impurity profiling of
drugs.
Impurities in new drug substances can be addressed from two perspectives,
(1) The chemical aspect which includes classification and identification of
impurities, report generation, listing of impurities in specifications, and a brief
discussion of analytical procedures,
(2) The safety aspect which includes, specific guidance for quantifying
impurities, present ,substantially at lower levels, in a drug substance used
inclinical studies.

ACCEPTANCE CRITERIA
The new drug substance specification should include, where applicable, the
following list of impurities:
1.Each specified identified impurity;
2.Each specified unidentified impurity;
3.Any unspecified impurity with an acceptance criterion of not more than
(≤) the identification threshold;
4.Total impurities.

Maximum Reporting Identification Qualification

Daily Dose Threshold Threshold Threshold
0.10% or 1.0 mg per day 0.15% or 1.0 mg per
≤ 2g/day 0.05% intake (whichever is day intake (whichever is
lower) lower)
> 2g/day 0.03% 0.05% 0.05%

Common Terms of Impurities:

Following terms are used by various regulatory bodies and ICH to describe
the impurities
1.Intermediate
2.Penultimate intermediate
3.By-products
4.Transformation products
5.Interaction products
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 6.Related products
7.Degradation products
1.Intermediate:
The compounds produced during synthesis of the desired material or as a
part of the route of synthesis.

2.Penultimate Intermediate:
It is the last compound in the synthesis chain prior to the production of the
final desired compound.
3.By-products:
The compound produced in the reaction other than the required
intermediates. They can occur through a variety of side reactions, such as
overreaction, incomplete reaction, demonization and rearrangement, unwanted
reactions between starting materials or intermediates with chemical reagents or
catalysts.
4.Transformation Products:
They are related to theorized and nontheorized products that can occur in a
reaction. They are similar to by-products except that more is known about these
reaction products.
5.Interaction Products:
These products formed either intentionally or unintentionally interaction
between various chemicals involved.
6.Related Products:
These are chemically similar to drug substance and may even possess
biological activity.
7.Degradation Products:
They are formed by the decomposition of active ingredient or other
material of interest by the effect of external factors like heat, light and moisture
Classification of Impurity:
United States Pharmacopoeia (USP)
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 According to USP impurities are classified into three sections:
1.Impurities in Official Articles
2.Ordinary Impurities
3.Organic Volatile Impurities
The ICH Terminology
According to ICH guidelines, impurities in drug substance produced by
chemical synthesis can be broadly classified into following three categories
1.Organic Impurities (Process and drug-related)
2.Inorganic Impurities (Reagent, ligands, catalysts)
3.Residual Solvents (Volatile solvents)
1. ORGANIC IMPURITIES
These types of impurities arise during the manufacturing process and/or
during storage of the drug substance. These include following sub-impurities.
Starting Materials or Intermediate Impurities
These types of impurities occur in almost every API unless a proper care is
taken in every step during the multistep synthesis of drug product. Although the
end products are always washed with solvents but there are chances of having the
residual of unreacted starting materials unless the manufacturers are very careful
about the impurities.
By-products
In synthetic organic chemistry, getting a single end product with complete
yield is very rare; there is always a chance of having by products along with
desired end product.
Degradation Products
Impurities can also be formed by degradation of the end product during
manufacturing of bulk drugs. This mainly occurs due to improper storage of
formulation.
Other Types of Organic Impurities
A. Synthesis Related Impurities

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 New chemical entity generated during synthetic process from raw material,
solvent, intermediate, by-product. During synthesis process, if impurity present
in trace or in significant amount in any of substance involved in reaction, that
ultimately result in final product contaminated with one or more unwanted
materials. Therefore, synthesis related impurity require upmost care during every
step involved in synthesis process to minimize level of impurity that can arise.
B. Formulation Related Impurities:
Drug substance subjected to variety of conditions that leads to its
degradation or other reactions. Solutions and suspensions are prone to
degradation due to hydrolysis. Water used in formulation contribute to not only
its impurity but also provide situation for hydrolysis and catalysis.
Factors Affecting On Formulation Related Impurities
a. Environment related
I.Exposed to adverse temperature: Substance which are labile to heat or in
tropical temperature lead to degradation of active constitute and formation of
impurity occurs. E.g. Vitamins are heat sensitive and its degradation lead to loss
in potency.
II.Exposed to light: Photosensitive material when exposed to light / UV light
undergo degradation which forms impurity.
III.Humidity: It can be detrimental to bulk powder and formulation containing
solid dosage form.
b. Formation of impurities on ageing:
Mutual interaction: Interaction between ingredients involved in formulation
leads to mutual interaction which causes impurity formation.
C. Functional Group Related Impurities
a)Ester hydrolysis: Drugs like aspirin, benzocaine, cefoxime, cocaine, ethyl
paraben undergo ester hydrolysis.
b)Hydrolysis: Commonly drugs like benzyl penicillin, barbital, and
chloramphenicol undergo hydrolysis.
c)Oxidative degradation: Drugs like hydrocortisone, methotrexate, heterocyclic
aromatic ring, nitroso/nitrile derivative.

125
d)Photolytic cleavage: Product exposed to light while manufacturing or storage
in hospital pending use or by consumer pending use.
e)Decarboxylation: Some dissolved carboxylic acid such as p-amino salicylic
acid loose CO2 when heated.
2. INORGANIC IMPURITIES
Inorganic impurities are also obtained from the manufacturing processes
which are used in bulk drug formulation. They are normally known and
identified.

a.Reagent, Ligands and Catalysts:

Rare chances of occurrence of these impurities. If during manufacturing
procedure is not followed properly will create a problem.
b.Heavy Metals:
Water is generally used in different manufacturing processes which act as
the main source of heavy metals, like Ar, Cd, Cr, Na, Mg, Mn, etc., where
acidification or acid hydrolysis takes place. By using demineralized water and
glass-lined reactors heavy metal impurities can be easily avoided.
c.Other Materials (Filter Aids, Charcoal):
The filters or filtering aids such as centrifuge bags are routinely used in the
bulk drugs manufacturing plants and in many cases, activated carbon is also used
which also act as a source of impurity. Therefore to avoid the contamination,
regular monitoring of fibers and black particles in the bulk drugs is essential.
3. RESIDUAL SOLVENTS
Residual solvents are organic or inorganic liquids used during the
manufacturing process. It is very difficult to remove these solvents completely by
the work-up process. Some solvent that are known to cause toxicity should be
avoided in the production of bulk drugs.
4. FORMULATION RELATED IMPURITIES(IMPURITIES IN DRUG
PRODUCTS):
Number of impurities in a drug product can arise out of inert ingredients
used to formulate a drug substance. In the process of formulation, a drug
126
substance is subjected to a variety of conditions that can lead to its degradation or
other deleterious reaction. Solutions and suspensions are potentially prone to
degradation due to hydrolysis. The water used in the formulation cannot only
contribute its own impurities; it can also provide a ripe situation for hydrolysis
and catalysis. Similar reactions are possible in other solvents that may be used.
The formulation related impurities can be classified as follows:
 Method related
 Environmental related
The primary environmental factors that can reduce stability include the following
I.Exposures to adverse temperatures
II.Light-especially UV light
III.Humidity
Dosage form related
I.Mutual interaction amongst ingredients
II.Functional group- related typical degradation
Ester hydrolysis
Hydrolysis
Oxidative degradation
Photolytic cleavage
Decarboxylation
Method related
A known impurity, 1-(2, 6-diclorophenyl) indolin-2-one is formed in the
production of a parenteral dosage form of diclofenac sodium if it is terminally
sterilized by autoclave. It was the condition of the autoclave method (ie, 123 +
2°C) that enforced the intramolecular cyclic reaction of diclofenac sodium
forming the indolinone derivative and sodium hydroxide. The formation of this
impurity has been found to depend on the initial pH of the formulation. The
concentration of the impurity in the resultant product in the ampoule exceeds the
limit of the raw material in the BP.
Environmental related
127
 The primary environmental factors that can reduce stability include the
following:
Exposures to adverse temperatures:
There are many API’s that are labile to heat or tropical temperatures. For
example, vitamins as drug substances are very heat-sensitive and degradation
frequently leads to loss of potency in vitamin products, especially in liquid
formulations.
Light-especially UV light:
Several studies have reported that ergometrine as well as methyl
ergometrine injection is unstable under tropical conditions such as light and heat
and a very low level of active ingredient was found in many field samples. In
only 50% of the marketed samples of ergometrine injections tested did the level
of active ingredient comply with the BP/USP limit of 90% to 110% of the stated
content. The custom-made injection of ergometrine (0.2mg/mL) showed almost
complete degradation when kept 42 hours in direct sunlight.
Humidity:
For hygroscopic products, humidity is considered detrimental to both bulk
powder and formulated solid dosage forms. Aspirin and ranitidine are classical
examples.
Dosage form related:
Although the pharmaceutical companies perform pre-formulation studies,
including a stability study, before marketing the products, sometimes the dosage
form factors that influence drug stability force the company to recall the product.
Fluocinonide Topical Solution USP, 0.05% (Teva Pharmaceuticals USA, Inc.,
Sellersville, Pennsylvania) in 60 mL bottles, was recalled in the United States
because of degradation/impurities leading to sub-potency. In general, liquid
dosage forms are very much susceptible to both degradation and microbiological
contamination. In this regard, water content, pH of the solution/suspension,
compatibility of anions and cations, mutual interactions of ingredients and the
primary container are critical factors.
Microbiological growth resulting from the growth of bacteria, fungi and
yeast in a humid and warm environment may result in oral liquid products that
are unusable for human consumption. Microbial contaminations may occur

128
during the shelf life and subsequent consumer-use of a multiple-dose product due
to inappropriate use of certain preservatives in the preparations or because of the
semi-permeable nature of primary containers.
ANALYTICAL METHOD DEVELOPMENT
New drug development requires meaningful and reliable analytical data to
be produced at various stages of the development.
a) Sample set selection for analytical method development
b) Screening of Chromatographic conditions and Phases, typically using the
linear solvent- strength model of gradient elution
c) Optimization of the method to fine-tune parameters related to ruggedness and
robustness
The impurities can be identified predominantly by following methods;
• Reference standard method
• Spectroscopic method
• Separation method
• Isolation method
• Characterization method
Characterization methods
Highly sophisticated instrumentation, such as MS attached to a GC or
HPLC, are inevitable tools in the identification of minor components (drugs,
impurities, degradation products, metabolites) in various matrices. For
characterization of impurities, different techniques are used; which are as
follows;
NMR
The ability of NMR to provide information regarding the specific bonding
structure and stereochemistry of molecules of pharmaceutical interest has made it
a powerful analytical instrument for structural elucidation. The ability of NMR-
based diffusion coefficient determination to distinguish between monomeric and
dimeric substances was validated using a standard mixture of authentic materials
containing both monomers and dimers. Unfortunately, NMR has traditionally
been used as a less sensitive method compared to other analytical techniques.
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Conventional sample requirements for NMR are on the order of 10 mg, as
compared with MS, which requires less than 1 mg.
MS
It has an increasingly significant impact on the pharmaceutical
development process over the past several decades. Advances in the design and
efficiency of the interfaces, that directly connect separation techniques with Mass
Spectrometers have afforded new opportunities for monitoring, characterizing,
and quantification of drug-related substances in active pharmaceutical
ingredients and pharmaceutical formulations. If single method fails to provide the
necessary selectivity, orthogonal coupling of chromatographic techniques such as
HPLC-TLC and HPLC-CE, or coupling of chromatographic separations with
information rich spectroscopic methods such as HPLC-MS or HPLC-NMR may
need to be contemplated, but hopefully only as a development tool rather than a
tool for routine QC use.
Hyphenated Methods:
• LC-MS-MS
• HPLC-DAD-MS
• HPLC-DAD-NMR-MS
• GC-MS
• LC-MS
An example of reverse-phase LC-MS analysis in gradient elution with two
distinct soft ionization techniques is the Atmospheric pressure ionization with
electrospray source (API-ESI) and the chemical ionization of d-allethrine.
A common goal for investigation of both process and product degradation-
related impurities is to determine which of the many potential impurities are, in
fact, produced in the manufacturing process and which occur under a given set of
storage conditions.
Goals of impurity investigations
Process-related impurities Degradation–related impurities

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Identify significant impurities Identify potential degradation
product
through stress testing and actual
degradation
products through stability studies.
Determine origin of impurities and Understand degradation pathway and
method for methods to minimize degradation
elimination or reduction

Establish a control system for Establish a control system for

impurities impurities
involving: involving:
1) Processing/manufacturing 1) Processing/manufacturing
conditions conditions
2) Suitable analytical methods/ 2) Suitable analytical methods/
specifications specifications
3) Long term storage conditions
including packaging
4) Formulation.
GUIDELINES FOR IMPURITY PROFILE:-
It is now getting important critical attention from regulatory authorities.
The different pharmacopoeias, such as the British Pharmacopoeia (BP), the
United States Pharmacopoeia (USP) and the Indian Pharmacopoeia (IP) are
slowly incorporating limits to allowable levels of impurities present in the APIs
or formulations. Also, the International Conference on Harmonization (ICH) has
published certain guidelines on impurities in drug substances, products and
residual solvents. There is a significant demand for the impurity reference
standards and the API reference standards for both regulatory authorities and
pharmaceutical companies. According to ICH guidelines on impurities in new
drug products, identification of impurities below 0.1% level is not considered to
be necessary, unless potential impurities are expected to be unusually potent or
toxic.

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GENERAL SCHEME FOR DRUG IMPURITY PROFILING:
APPLICATIONS
Numerous applications have been sought in the areas of drug designing and
in monitoring quality, stability, and safety of pharmaceutical compounds,
whether produced synthetically, extracted from natural products or produced by
recombinant methods. The applications include alkaloids, amines, amino acids,
analgesics, antibacterials, anticonvulsants, antidepressant, tranquilizers,
antineoplastic agents, local anesthetics, macromolecules, steroids, miscellaneous.
STABILITY TESTING:PHOTOSTABILITY TESTING OF NEW DRUG
SUBSTANCES AND PRODUCTS Q1B
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1. GENERAL
The ICH Harmonized Tripartite Guideline covering the Stability Testing of
New Drug Substances and Products (hereafter referred to as the Parent
Guideline) notes that light testing should be an integral part of stress testing. This
document is an annex to the Parent Guideline and addresses the
recommendations for photostability testing.
A. Preamble
The intrinsic photostability characteristics of new drug substances and
products should be evaluated to demonstrate that, as appropriate, light exposure
does not result in unacceptable change. Normally, photostability testing is carried
out on a single batch of material selected as described under Selection of Batches
in the Parent Guideline. Under some circumstances these studies should be
repeated if certain variations and changes are made to the product (e.g.,
formulation, packaging). Whether these studies should be repeated depends on
the photostability characteristics determined at the time of initial filing and the
type of variation and/or change made.
The guideline primarily addresses the generation of photostability
information for submission in Registration Applications for new molecular
entities and associated drug products. The guideline does not cover the
photostability of drugs after administration (i.e. under conditions of use) and
those applications not covered by the Parent Guideline. Alternative approaches
may be used if they are scientifically sound and justification is provided.
A systematic approach to photostability testing is recommended covering,
as appropriate, studies such as:
i) Tests on the drug substance;
ii) Tests on the exposed drug product outside of the immediate pack;
and if necessary ;
iii) Tests on the drug product in the immediate pack;
and if necessary ;
iv) Tests on the drug product in the marketing pack.
The extent of drug product testing should be established by assessing
whether or not acceptable change has occurred at the end of the light exposure
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testing as described in the Decision Flow Chart for Photostability Testing of
Drug Products. Acceptable change is change within limits justified by the
applicant.
The formal labeling requirements for photolabile drug substances and drug
products are established by national/regional requirements.
B. Light Sources
The light sources described below may be used for photostability testing.
The applicant should either maintain an appropriate control of temperature to
minimize the effect of localized temperature changes or include a dark control in
the same environment unless otherwise justified. For both options 1 and 2, a
pharmaceutical manufacturer/applicant may rely on the spectral distribution
specification of the light source manufacturer.
Option 1
Any light source that is designed to produce an output similar to the
D65/ID65 emission standard such as an artificial daylight fluorescent lamp
combining visible and ultraviolet (UV) outputs, xenon, or metal halide lamp.
D65 is the internationally recognized standard for outdoor daylight as defined in
ISO 10977 (1993). ID65 is the equivalent indoor indirect daylight standard. For a
light source emitting significant radiation below 320 nm, an appropriate filter(s)
may be fitted to eliminate such radiation.
Option 2
For option 2 the same sample should be exposed to both the cool white
fluorescent and near ultraviolet lamp.
1. A cool white fluorescent lamp designed to produce an output
similar to that specified in ISO 10977(1993) ; and
2. A near UV fluorescent lamp having a spectral distribution from 320
nm to 400 nm with a maximum energy emission between 350 nm and 370
nm; a significant proportion of UV should be in both bands of 320 to 360
nm and 360 to 400 nm.
C. Procedure
For confirmatory studies, samples should be exposed to light providing an
overall illumination of not less than 1.2 million lux hours and an integrated near

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ultraviolet energy of not less than 200 watt hours/square meter to allow direct
comparisons to be made between the drug substance and drug product.
Samples may be exposed side-by-side with a validated chemical
actinometric system to ensure the specified light exposure is obtained, or for the
appropriate duration of time when conditions have been monitored using
calibrated radiometers/lux meters. An example of an actinometric procedure is
provided in the Annex.
If protected samples (e.g., wrapped in aluminum foil) are used as dark
controls to evaluate the contribution of thermally induced change to the total
observed change, these should be placed alongside the authentic sample.

2. DRUG SUBSTANCE
For drug substances, photostability testing should consist of two parts:
forced degradation testing and confirmatory testing.

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 The purpose of forced degradation testing studies is to evaluate the overall
photosensitivity of the material for method development purposes and/or
degradation pathway elucidation. This testing may involve the drug substance
alone and/or in simple solutions/suspensions to validate the analytical
procedures. In these studies, the samples should be in chemically inert and
transparent containers. In these forced degradation studies, a variety of exposure
conditions may be used, depending on the photosensitivity of the drug substance
involved and the intensity of the light sources used. For development and
validation purposes it is appropriate to limit exposure and end the studies if
extensive decomposition occurs. For photostable materials, studies may be
terminated after an appropriate exposure level has been used. The design of these
experiments is left to the applicant’s discretion although the exposure levels used
should be justified.
Under forcing conditions, decomposition products may be observed that are
unlikely to be formed under the conditions used for confirmatory studies. This
information may be useful in developing and validating suitable analytical
methods. If in practice it has been demonstrated they are not formed in the
confirmatory studies, these degradation products need not be further examined.
Confirmatory studies should then be undertaken to provide the information
necessary for handling, packaging, and labeling (see section I.C., Procedure, and
II.A., Presentation, for information on the design of these studies).
Normally, only one batch of drug substance is tested during the
development phase, and then the photostability characteristics should be
confirmed on a single batch selected as described in the Parent Guideline if the
drug is clearly photostable or photolabile. If the results of the confirmatory study
are equivocal, testing of up to two additional batches should be conducted.
Samples should be selected as described in the Parent Guideline.
A. Presentation of Samples
Care should be taken to ensure that the physical characteristics of the
samples under test are taken into account and efforts should be made, such as
cooling and/or placing the samples in sealed containers, to ensure that the effects
of the changes in physical states such as sublimation, evaporation or melting are
minimized. All such precautions should be chosen to provide minimal
interference with the exposure of samples under test. Possible interactions
between the samples and any material used for containers or for general
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protection of the sample, should also be considered and eliminated wherever not
relevant to the test being carried out.
As a direct challenge for samples of solid drug substances, an appropriate
amount of sample should be taken and placed in a suitable glass or plastic dish
and protected with a suitable transparent cover if considered necessary. Solid
drug substances should be spread across the container to give a thickness of
typically not more than 3 millimeters. Drug substances that are liquids should be
exposed in chemically inert and transparent containers.
B. Analysis of Samples
At the end of the exposure period, the samples should be examined for any
changes in physical properties (e.g., appearance, clarity, or color of solution) and
for assay and degradants by a method suitably validated for products likely to
arise from photochemical degradation processes.
Where solid drug substance samples are involved, sampling should ensure
that a representative portion is used in individual tests. Similar sampling
considerations, such as homogenization of the entire sample, apply to other
materials that may not be homogeneous after exposure. The analysis of the
exposed sample should be performed concomitantly with that of any protected
samples used as dark controls if these are used in the test.
C. Judgement of Results
The forced degradation studies should be designed to provide suitable
information to develop and validate test methods for the confirmatory studies.
These test methods should be capable of resolving and detecting photolytic
degradants that appear during the confirmatory studies. When evaluating the
results of these studies, it is important to recognize that they form part of the
stress testing and are not therefore designed to establish qualitative or
quantitative limits for change.
The confirmatory studies should identify precautionary measures needed in
manufacturing or in formulation of the drug product, and if light resistant
packaging is needed. When evaluating the results of confirmatory studies to
determine whether change due to exposure to light is acceptable, it is important
to consider the results from other formal stability studies in order to assure that
the drug will be within justified limits at time of use (see the relevant ICH
Stability and Impurity Guidelines).
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3. DRUG PRODUCT
Normally, the studies on drug products should be carried out in a sequential
manner starting with testing the fully exposed product then progressing as
necessary to the product in the immediate pack and then in the marketing pack.
Testing should progress until the results demonstrate that the drug product is
adequately protected from exposure to light. The drug product should be exposed
to the light conditions described under the procedure in section I.C.
Normally, only one batch of drug product is tested during the development
phase, and then the photostability characteristics should be confirmed on a single
batch selected as described in the Parent Guideline if the product is clearly
photostable or photolabile. If the results of the confirmatory study are equivocal,
testing of up to two additional batches should be conducted.
For some products where it has been demonstrated that the immediate pack
is completely impenetrable to light, such as aluminium tubes or cans, testing
should normally only be conducted on directly exposed drug product.
It may be appropriate to test certain products such as infusion liquids,
dermal creams, etc., to support their photostability in-use. The extent of this
testing should depend on and relate to the directions for use, and is left to the
applicant’s discretion.
The analytical procedures used should be suitably validated.
A. Presentation of Samples
Care should be taken to ensure that the physical characteristics of the
samples under test are taken into account and efforts, such as cooling and/or
placing the samples in sealed containers, should be made to ensure that the
effects of the changes in physical states are minimized, such as sublimation,
evaporation, or melting. All such precautions should be chosen to provide a
minimal interference with the irradiation of samples under test. Possible
interactions between the samples and any material used for containers or for
general protection of the sample should also be considered and eliminated
wherever not relevant to the test being carried out.
Where practicable when testing samples of the drug product outside of the
primary pack, these should be presented in a way similar to the conditions

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mentioned for the drug substance. The samples should be positioned to provide
maximum area of exposure to the light source. For example, tablets, capsules,
etc., should be spread in a single layer.
If direct exposure is not practical (e.g., due to oxidation of a product), the
sample should be placed in a suitable protective inert transparent container (e.g.,
quartz).
If testing of the drug product in the immediate container or as marketed is
needed, the samples should be placed horizontally or transversely with respect to
the light source, whichever provides for the most uniform exposure of the
samples. Some adjustment of testing conditions may have to be made when
testing large volume containers (e.g., dispensing packs).
B. Analysis of Samples
At the end of the exposure period, the samples should be examined for any
changes in physical properties (e.g., appearance, clarity or color of solution,
dissolution/disintegration for dosage forms such as capsules, etc.) and for assay
and degradants by a method suitably validated for products likely to arise from
photochemical degradation processes.
When powder samples are involved, sampling should ensure that a
representative portion is used in individual tests. For solid oral dosage form
products, testing should be conducted on an appropriately sized composite of, for
example, 20 tablets or capsules. Similar sampling considerations, such as
homogenization or solubilization of the entire sample, apply to other materials
that may not be homogeneous after exposure (e.g., creams, ointments,
suspensions, etc.). The analysis of the exposed sample should be performed
concomitantly with that of any protected samples used as dark controls if these
are used in the test.
C. Judgement of Results
Depending on the extent of change special labeling or packaging may be
needed to mitigate exposure to light. When evaluating the results of
photostability studies to determine whether change due to exposure to light is
acceptable, it is important to consider the results obtained from other formal
stability studies in order to assure that the product will be within proposed
specifications during the shelf life (see the relevant ICH Stability and Impurity
Guidelines).
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4.GLOSSARY
Immediate (primary) pack is that constituent of the packaging that is in
direct contact with the drug substance or drug product, and includes any
appropriate label.
Marketing pack is the combination of immediate pack and other secondary
packaging such as a carton.
Forced degradation testing studies are those undertaken to degrade the
sample deliberately. These studies, which may be undertaken in the development
phase normally on the drug substances, are used to evaluate the overall
photosensitivity of the material for method development purposes and/or
degradation pathway elucidation.
Confirmatory studies are those undertaken to establish photostability
characteristics under standardized conditions. These studies are used to identify
precautionary measures needed in manufacturing or formulation and whether
light resistant packaging and/or special labeling is needed to mitigate exposure to
light. For the confirmatory studies, the batch(es) should be selected according to
batch selection for long-term and accelerated testings which is described in the
Parent Guideline.
QUALITY OF BIOTECHNOLOGICAL PRODUCTS:
STABILITY TESTING OF BIOTECHNOLOGICAL/BIOLOGICAL
PRODUCTS
1. PREAMBLE
The guidance stated in the ICH harmonised tripartite guideline “Stability
Testing of New Drug Substances and Products” (27 October 1993) applies in
general to biotechnological/biological products. However,
biotechnological/biological products do have distinguishing characteristics to
which consideration should be given in any well-defined testing program
designed to confirm their stability during the intended storage period. For such
products, in which the active components are typically proteins and/or
polypeptides, maintenance of molecular conformation and, hence of biological
activity, is dependent on noncovalent as well as covalent forces. The products are
particularly sensitive to environmental factors such as temperature changes,
oxidation, light, ionic content, and shear. In order to ensure maintenance of

140
biological activity and to avoid degradation, stringent conditions for their storage
are usually necessary.
The evaluation of stability may necessitate complex analytical
methodologies. Assays for biological activity, where applicable, should be part of
the pivotal stability studies. Appropriate physicochemical, biochemical and
immunochemical methods for the analysis of the molecular entity and the
quantitative detection of degradation products should also be part of the stability
program whenever purity and molecular characteristics of the product permit use
of these methodologies.
With the above concerns in mind, the applicant should develop the proper
supporting stability data for a biotechnological/biological product and consider
many external conditions which can affect the product’s potency, purity and
quality. Primary data to support a requested storage period for either drug
substance or drug product should be based on long-term, real-time, real-condition
stability studies. Thus, the development of a proper long-term stability program
becomes critical to the successful development of a commercial product. The
purpose of this document is to give guidance to applicants regarding the type of
stability studies that should be provided in support of marketing applications. It is
understood that during the review and evaluation process, continuing updates of
initial stability data may occur.
2. SCOPE OF THE ANNEX
The guidance stated in this annex applies to well-characterised proteins and
polypeptides, their derivatives and products of which they are components, and
which are isolated from tissues, body fluids, cell cultures, or produced using
rDNA technology. Thus, the document covers the generation and submission of
stability data for products such as cytokines (interferons, interleukins, colony-
stimulating factors, tumour necrosis factors), erythropoietins, plasminogen
activators, blood plasma factors, growth hormones and growth factors, insulins,
monoclonal antibodies, and vaccines consisting of well-characterised proteins or
polypeptides. In addition, the guidance outlined in the following sections may
apply to other types of products, such as conventional vaccines, after consultation
with the appropriate regulatory authorities. The document does not cover
antibiotics, allergenic extracts, heparins, vitamins, whole blood, or cellular blood
components.
3. TERMINOLOGY
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 For the basic terms used in this annex the reader is referred to the
"Glossary" in the ICH harmonised tripartite guideline "Stability Testing of New
Drug Substances and Products" (27 October 1993). However, since
manufacturers of biotechnological/biological products sometimes use traditional
terminology, traditional terms are specified in parentheses to assist the reader. A
supplemental glossary is also included that explains certain terms used in the
production of biotechnological/biological products.
4. SELECTION OF BATCHES
4.1. Drug Substance (Bulk Material)
Where bulk material is to be stored after manufacture but prior to
formulation and final manufacturing, stability data should be provided on at least
3 batches for which manufacture and storage are representative of the
manufacturing scale of production. A minimum of 6 months stability data at the
time of submission should be submitted in cases where storage periods greater
than 6 months are requested. For drug substances with storage periods of less
than 6 months, the minimum amount of stability data in the initial submission
should be determined on a case-by-case basis. Data from pilot-plant scale batches
of drug substance produced at a reduced scale of fermentation and purification
may be provided at the time the dossier is submitted to the regulatory agencies
with a commitment to place the first 3 manufacturing scale batches into the long-
term stability program after approval.
The quality of the batches of drug substance placed into the stability
program should be representative of the quality of the material used in preclinical
and clinical studies and of the quality of the material to be made at
manufacturing scale. In addition, the drug substance (bulk material) made at
pilot-plant scale should be produced by a process and stored under conditions
representative of that used for the manufacturing scale. The drug substance
entered into the stability program should be stored in containers which properly
represent the actual holding containers used during manufacture. Containers of
reduced size may be acceptable for drug substance stability testing provided that
they are constructed of the same material and use the same type of
container/closure system that is intended to be used during manufacture.
4.2. Intermediates
During manufacture of biotechnological/biological products, the quality
and control of certain intermediates may be critical to the production of the final
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product. In general, the manufacturer should identify intermediates and generate
in-house data and process limits that assure their stability within the bounds of
the developed process. While the use of pilot-plant scale data is permissible, the
manufacturer should establish the suitability of such data using the
manufacturing scale process.
4.3. Drug Product (Final Container Product)
Stability information should be provided on at least 3 batches of final
container product representative of that which will be used at manufacturing
scale. Where possible, batches of final container product included in stability
testing should be derived from different batches of bulk material. A minimum of
6 months data at the time of submission should be submitted in cases where
storage periods greater than 6 months are requested. For drug products with
storage periods of less than 6 months, the minimum amount of stability data in
the initial submission should be determined on a case-by-case basis. Product
expiration dating will be based upon the actual data submitted in support of the
application. Since dating is based upon the real-time/real-temperature data
submitted for review, continuing updates of initial stability data should occur
during the review and evaluation process. The quality of the final container
product placed on stability studies should be representative of the quality of the
material used in the preclinical and clinical studies. Data from pilot-plant scale
batches of drug product may be provided at the time the dossier is submitted to
the regulatory agencies with a commitment to place the first 3 manufacturing
scale batches into the long term stability program after approval. Where pilot-
plant scale batches were submitted to establish the dating for a product and, in
the event that product produced at manufacturing scale does not meet those long-
term stability specifications throughout the dating period or is not representative
of the material used in preclinical and clinical studies, the applicant should notify
the appropriate regulatory authorities to determine a suitable course of action.
4.4. Sample Selection
Where one product is distributed in batches differing in fill volume (e.g., 1
millilitre (ml), 2 ml, or 10 ml), unitage (e.g., 10 units, 20 units, or 50 units), or
mass (e.g., 1 milligram (mg), 2 mg, or 5 mg) samples to be entered into the
stability program may be selected on the basis of a matrix system and/or by
bracketing.

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 Matrixing, i.e., the statistical design of a stability study in which different
fractions of samples are tested at different sampling points, should only be
applied when appropriate documentation is provided that confirms that the
stability of the samples tested represents the stability of all samples. The
differences in the samples for the same drug product should be identified as, for
example, covering different batches, different strengths, different sizes of the
same closure and possibly, in some cases, different container/closure systems.
Matrixing should not be applied to samples with differences that may affect
stability, such as different strengths and different containers/closures, where it
cannot be confirmed that the products respond similarly under storage conditions.
Where the same strength and exact container/closure system is used for 3 or
more fill contents, the manufacturer may elect to place only the smallest and
largest container size into the stability program, i.e., bracketing. The design of a
protocol that incorporates bracketing assumes that the stability of the
intermediate condition samples are represented by those at the extremes. In
certain cases, data may be needed to demonstrate that all samples are properly
represented by data collected for the extremes.
5. STABILITY-INDICATING PROFILE
On the whole, there is no single stability-indicating assay or parameter that
profiles the stability characteristics of a biotechnological/biological product.
Consequently, the manufacturer should propose a stability-indicating profile that
provides assurance that changes in the identity, purity and potency of the product
will be detected.
At the time of submission, applicants should have validated the methods
that comprise the stability-indicating profile and the data should be available for
review. The determination of which tests should be included will be product-
specific. The items emphasised in the following subsections are not intended to
be all-inclusive, but represent product characteristics that should typically be
documented to adequately demonstrate product stability.
5.1. Protocol
The dossier accompanying the application for marketing authorisation
should include a detailed protocol for the assessment of the stability of both drug
substance and drug product in support of the proposed storage conditions and
expiration dating periods. The protocol should include all necessary information
which demonstrates the stability of the biotechnological/biological product
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throughout the proposed expiration dating period including, for example, well-
defined specifications and test intervals. The statistical methods that should be
used are described in the tripartite guideline on stability.
5.2. Potency
When the intended use of a product is linked to a definable and measurable
biological activity, testing for potency should be part of the stability studies. For
the purpose of stability testing of the products described in this guideline,
potency is the specific ability or capacity of a product to achieve its intended
effect. It is based on the measurement of some attribute of the product and is
determined by a suitable quantitative method. In general, potencies of
biotechnological/biological products tested by different laboratories can be
compared in a meaningful way only if expressed in relation to that of an
appropriate reference material. For that purpose, a reference material calibrated
directly or indirectly against the corresponding national or international reference
material should be included in the assay.
Potency studies should be performed at appropriate intervals as defined in
the stability protocol and the results should be reported in units of biological
activity calibrated, whenever possible, against nationally or internationally
recognised standard. Where no national or international reference standards exist,
the assay results may be reported in in-house derived units using a characterised
reference material.
In some biotechnological/biological products, potency is dependent upon
the conjugation of the active ingredient(s) to a second moiety or binding to an
adjuvant. Dissociation of the active ingredient(s) from the carrier used in
conjugates or adjuvants should be examined in real-time/real-temperature studies
(including conditions encountered during shipment). The assessment of the
stability of such products may be difficult since, in some cases, in vitro tests for
biological activity and physicochemical characterisation are impractical or
provide inaccurate results. Appropriate strategies (e.g., testing the product prior
to conjugation/binding, assessing the release of the active compound from the
second moiety, in vivo assays) or the use of an appropriate surrogate test should
be considered to overcome the inadequacies of in vitro testing.
5.3. Purity and Molecular Characterisation
For the purpose of stability testing of the products described in this
guideline, purity is a relative term. Due to the effect of glycosylation,
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deamidation, or other heterogeneities, the absolute purity of a
biotechnological/biological product is extremely difficult to determine. Thus, the
purity of a biotechnological/biological product should be typically assessed by
more than one method and the purity value derived is method-dependent. For the
purpose of stability testing, tests for purity should focus on methods for
determination of degradation products.
The degree of purity, as well as individual and total amounts of degradation
products of the biotechnological/biological product entered into the stability
studies, should be reported and documented whenever possible. Limits of
acceptable degradation should be derived from the analytical profiles of batches
of the drug substance and drug product used in the preclinical and clinical
studies.
The use of relevant physicochemical, biochemical and immunochemical
analytical methodologies should permit a comprehensive characterisation of the
drug substance and/or drug product (e.g., molecular size, charge, hydrophobicity)
and the accurate detection of degradation changes that may result from
deamidation, oxidation, sulfoxidation, aggregation or fragmentation during
storage. As examples, methods that may contribute to this include electrophoresis
(SDS-PAGE, immunoelectrophoresis, Western blot, isoelectrofocusing), high-
resolution chromatography (e.g., reversed-phase chromatography, gel filtration,
ion exchange, affinity chromatography), and peptide mapping.
Wherever significant qualitative or quantitative changes indicative of
degradation product formation are detected during long-term, accelerated and/or
stress stability studies, consideration should be given to potential hazards and to
the need for characterisation and quantification of degradation products within
the long-term stability program. Acceptable limits should be proposed and
justified, taking into account the levels observed in material used in preclinical
and clinical studies.
For substances that cannot be properly characterised or products for which
an exact analysis of the purity cannot be determined through routine analytical
methods, the applicant should propose and justify alternative testing procedures.
5.4. Other Product Characteristics
The following product characteristics, though not specifically relating to
biotechnological/biological products, should be monitored and reported for the
drug product in its final container:
146
 Visual appearance of the product (colour and opacity for
solutions/suspensions; colour, texture and dissolution time for powders), visible
particulates in solutions or after the reconstitution of powders or lyophilised
cakes, pH, and moisture level of powders and lyophilised products.
Sterility testing or alternatives (e.g., container/closure integrity testing)
should be performed at a minimum initially and at the end of the proposed shelf-
life.
Additives (e.g., stabilisers, preservatives) or excipients may degrade during
the dating period of the drug product. If there is any indication during
preliminary stability studies that reaction or degradation of such materials
adversely affect the quality of the drug product, these items may need to be
monitored during the stability program.
The container/closure has the potential to adversely affect the product and
should be carefully evaluated (see below).
6. STORAGE CONDITIONS
6.1. Temperature
Since most finished biotechnological/biological products need precisely
defined storage temperatures, the storage conditions for the real-time/real-
temperature stability studies may be confined to the proposed storage
temperature.

6.2. Humidity
Biotechnological/biological products are generally distributed in containers
protecting them against humidity. Therefore, where it can be demonstrated that
the proposed containers (and conditions of storage) afford sufficient protection
against high and low humidity, stability tests at different relative humidities can
usually be omitted. Where humidity-protecting containers are not used,
appropriate stability data should be provided.
6.3. Accelerated and Stress Conditions
As previously noted, the expiration dating should be based on real-
time/real-temperature data. However, it is strongly suggested that studies be
conducted on the drug substance and drug product under accelerated and stress
conditions. Studies under accelerated conditions may provide useful support data
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for establishing the expiration date, provide product stability information for
future product development (e.g., preliminary assessment of proposed
manufacturing changes such as change in formulation, scale-up), assist in
validation of analytical methods for the stability program, or generate
information which may help elucidate the degradation profile of the drug
substance or drug product. Studies under stress conditions may be useful in
determining whether accidental exposures to conditions other than those
proposed (e.g., during transportation) are deleterious to the product and also for
evaluating which specific test parameters may be the best indicators of product
stability. Studies of the exposure of the drug substance or drug product to
extreme conditions may help to reveal patterns of degradation; if so, such
changes should be monitored under proposed storage conditions. While the
tripartite guideline on stability describes the conditions of the accelerated and
stress study, the applicant should note that those conditions may not be
appropriate for biotechnological/biological products. Conditions should be
carefully selected on a case-by-case basis.
6.4. Light
Applicants should consult the appropriate regulatory authorities on a case-
by-case basis to determine guidance for testing.
6.5. Container/Closure
Changes in the quality of the product may occur due to the interactions
between the formulated biotechnological/biological product and
container/closure. Where the lack of interactions cannot be excluded in liquid
products (other than sealed ampoules), stability studies should include samples
maintained in the inverted or horizontal position (i.e., in contact with the
closure), as well as in the upright position, to determine the effects of the closure
on product quality. Data should be supplied for all different container/closure
combinations that will be marketed.
In addition to the standard data necessary for a conventional single-use vial,
the applicant should demonstrate that the closure used with a multiple-dose vial
is capable of withstanding the conditions of repeated insertions and withdrawals
so that the product retains its full potency, purity, and quality for the maximum
period specified in the instructions-for-use on containers, packages, and/or
package inserts. Such labelling should be in accordance with relevant
national/regional requirements.
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6.6. Stability after Reconstitution of Freeze-Dried Product
The stability of freeze-dried products after their reconstitution should be
demonstrated for the conditions and the maximum storage period specified on
containers, packages, and/or package inserts. Such labelling should be in
accordance with relevant national/regional requirements.
7. TESTING FREQUENCY
The shelf-lives of biotechnological/biological products may vary from days
to several years. Thus, it is difficult to draft uniform guidelines regarding the
stability study duration and testing frequency that would be applicable to all
types of biotechnological/biological products. With only a few exceptions,
however, the shelf-lives for existing products and potential future products will
be within the range of 0.5 to 5 years. Therefore, the guidance is based upon
expected shelf-lives in that range. This takes into account the fact that
degradation of biotechnological/biological products may not be governed by the
same factors during different intervals of a long storage period.
When shelf-lives of 1 year or less are proposed, the real-time stability
studies should be conducted monthly for the first 3 months and at 3 month
intervals thereafter.
For products with proposed shelf-lives of greater than 1 year, the studies
should be conducted every 3 months during the first year of storage, every 6
months during the second year, and annually thereafter.
While the testing intervals listed above may be appropriate in the pre-
approval or pre-licence stage, reduced testing may be appropriate after approval
or licensure where data are available that demonstrate adequate stability. Where
data exist that indicate the stability of a product is not compromised, the
applicant is encouraged to submit a protocol which supports elimination of
specific test intervals (e.g., 9 month testing) for post-approval/post-licensure,
long-term studies.
8. SPECIFICATIONS
Although biotechnological/biological products may be subject to significant
losses of activity, physicochemical changes, or degradation during storage,
international and national regulations have provided little guidance with respect
to distinct release and end of shelf-life specifications. Recommendations for
maximum acceptable losses of activity, limits for physicochemical changes, or
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degradation during the proposed shelf-life have not been developed for
individual types or groups of biotechnological/biological products but are
considered on a case-by-case basis. Each product should retain its specifications
within established limits for safety, purity, and potency throughout its proposed
shelf-life. These specifications and limits should be derived from all available
information using the appropriate statistical methods. The use of different
specifications for release and expiration should be supported by sufficient data to
demonstrate that clinical performance is not affected as discussed in the tripartite
guideline on stability.
9. LABELLING
For most biotechnological/biological drug substances and drug products,
precisely defined storage temperatures are recommended. Specific
recommendations should be stated, particularly for drug substances and drug
products that cannot tolerate freezing. These conditions, and where appropriate,
recommendations for protection against light and/or humidity, should appear on
containers, packages, and/or package inserts. Such labelling should be in
accordance with relevant national/regional requirements.
10. GLOSSARY
Conjugated Product
A conjugated product is made up of an active ingredient (for example,
peptide, carbohydrate) bound covalently or noncovalently to a carrier (for
example, protein, peptide, inorganic mineral) with the objective of improving the
efficacy or stability of the product.
Degradation Product
A molecule resulting from a change in the drug substance (bulk material)
brought about over time. For the purpose of stability testing of the products
described in this guideline, such changes could occur as a result of processing or
storage (e.g., by deamidation, oxidation, aggregation, proteolysis). For
biotechnological/biological products some degradation products may be active.
Impurity
Any component of the drug substance (bulk material) or drug product (final
container product) which is not the chemical entity defined as the drug substance,
an excipient, or other additives to the drug product.

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Intermediate
For biotechnological/biological products, a material produced during a
manufacturing process which is not the drug substance or the drug product but
whose manufacture is critical to the successful production of the drug substance
or the drug product. Generally, an intermediate will be quantifiable and
specifications will be established to determine the successful completion of the
manufacturing step prior to continuation of the manufacturing process. This
includes material which may undergo further molecular modification or be held
for an extended period of time prior to further processing.
Pilot-Plant Scale
The production of the drug substance or drug product by a procedure fully
representative of and simulating that to be applied at manufacturing scale. The
methods of cell expansion, harvest, and product purification should be identical
except for the scale of production.

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 UNIT-IV
STABILITY TESTING OF PHYTOPHARMACEUTICALS
INTRODUCTION
Herbal drugs referred as plants materials or herbalism, involves the use of
whole plants or parts of plants, to treat injuries or illnesses. Herbal drugs are the
use of therapeutic herbs to prevent and treat diseases and ailments or to support
health and healing. These are drugs or preparations made from a plant or plants
and used for any of such purposes. Herbal drugs are the oldest form of health
care known to mankind. There are many herbal products offered that assert to
treat the symptoms of a broad range of problems, from depression to cold and
flu. World Health Organization (WHO) has distinct herbal drugs as complete,
labeled medicinal products that have vigorous ingredients, aerial or secretive
parts of the plant or other plant material or combinations. World Health
Organization has set precise guidelines for the evaluation of the safety, efficacy,
and quality of herbal medicines. WHO estimates that 80% of the world
populations currently use herbal drugs for major healthcare. Exceptionally, in
some countries herbal drugs may also enclose by tradition, natural organic or
inorganic active constituents which
are not of plant source. The herbal drug is a chief constituent in traditional
medicine and a common constituent in ayurvedic, homoeopathic, naturopathic
and other medicine systems. Herbs are usually considered as safe since they
belong to natural sources. The use of herbal drugs due to toxicity and side effects
of allopathic medicines has led to rapid increase in the number of herbal drug
manufacturers. For the past few decades, herbal drugs have been more and more
consumed by the people with no prescription. Seeds, leaves, stems, bark, roots,
flowers, and extracts of all of these have been used in herbal drugs over the
millennia of their use. Herbal products have reached extensive adequacy as
beneficial agents like antimicrobial, antidiabetic, antifertility, antiageing,
antiarthritic, sedative, antidepressant, antianxiety, antispasmodic, analgesic, anti-
inflammatory, anti-HIV, vasodilatory, hepatoprotective, treatment of cirrhosis,
asthma, acne, impotence, menopause, migraine, gall stones, chronic fatigue,
Alzheimer's disease, and memory enhancing activities. Herbal drugs have been
recognized for approximately 4000 years. These drugs have survived real world
testing and thousands of years of human testing. Some drugs have been

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discontinued due to their toxicity, while others have been modified or combined
with additional herbs to counterbalance side effects.
ADVANTAGES OF HERBAL DRUGS
Low/Minimum cost
Potency and efficiency
Enhanced tolerance
More protection
Fewer side-effects
Complete accessibility
Recyclable
DISADVANTAGES OF HERBAL DRUGS
Not able to cure rapid sickness and accidents
Risk with self-dosing
Complexity in standardizations
HERBAL- DRUG INTERACTIONS:
Herbs are often administered in combination with therapeutic drugs, raising
the potential for herb-drug interactions. Cases have been published reporting
enhanced anticoagulation and bleeding when patients on long-term warfarin
therapy also took Salvia miltiorrhiza (danshen). Allium sativum (garlic)
decreased the area under the plasma concentration-time curve (AUC) and
maximum plasma concentration of saquinavir, but not ritonavir and paracetamol
(acetaminophen), in volunteers. A. sativum increased the clotting time and
international normalised ratio of warfarin and caused hypoglycemia when taken
with chlorpropamide. Ginkgo biloba (ginkgo) caused bleeding when combined
with warfarin or aspirin (acetylsalicylic acid), raised blood pressure when
combined with a thiazide diuretic and even caused coma when combined with
trazodone in patients. Panax ginseng (ginseng) reduced the blood concentrations
of alcohol (ethanol) and warfarin, and induced mania when used concomitantly
with phenelzine, but ginseng increased the efficacy of influenza vaccination.
Scutellaria baicalensis (huangqin) ameliorated irinotecan-induced gastrointestinal
toxicity in cancer patients.Piper methysticum (kava) increased the 'off' periods in
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patients with parkinsonism taking levodopa and induced a semicomatose state
when given concomitantly with alprazolam. Kava enhanced the hypnotic effect
of alcohol in mice, but this was not observed in humans. Silybum marianum
(milk thistle) decreased the trough concentrations of indinavir in humans.
Piperine from black (Piper nigrum Linn) and long (P. longum Linn) peppers
increased the AUC of phenytoin, propranolol and theophylline in healthy
volunteers and plasma concentrations of rifamipicin (rifampin) in patients with
pulmonary tuberculosis. Interactions between herbal medicines and prescribed
drugs can occur and may lead to serious clinical consequences. There are other
theoretical interactions indicated by preclinical data. Both pharmacokinetic
and/or pharmacodynamic
mechanisms have been considered to play a role in these interactions, although
the underlying mechanisms for the altered drug effects and/or concentrations by
concomitant herbal medicinesare yet to be determined. The clinical importance
of herb-drug interactions depends on many factors associated with the particular
herb, drug, and patient. Herbs should be appropriately labeled to alert consumers
to potential interactions when concomitantly used with drugs, and to recommend
a consultation with their general practitioners.
STANDARDIZATION OF HERBAL DRUGS
Standardized herbal products of consistent quality and containing well-
defined constituents are required for reliable clinical trials and to provide
consistent beneficial therapeutic effects. Pharmacological properties of an herbal
formulation depend on phytochemical constituents present therein. Development
of authentic analytical methods which can reliably profile the phytochemical
composition, including quantitative analyses of marker/bioactive compounds and
other major constituents, is a major challenge to scientists. The resurgence of
interest and the growing market of herbal medicinal products necessitate strong
commitment by the stakeholders to safeguard the consumer and the industry.
Standardization is the first step for the establishment of a consistent biological
activity, a consistent chemical profile, or simply a quality assurance program for
production and manufacturing. Therefore, the EU has defined three categories of
herbal products:
Those containing constituents (single compounds or families of compounds)
with known and experienced therapeutic activity that are deemed solely
responsible for clinical efficacy.
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 Those containing chemically defined constituents possessing relevant
pharmacological properties which are likely to contribute to the clinical efficacy.
Those in which no constituents have been identified as being responsible for
the therapeutic activity.
Standardization, as defined in the text for guidance on the quality of herbal
medicinal products, means adjusting the herbal drug preparation to a defined
content of a constituent or group of substances with known therapeutic activity.
The European Medicines Agency (EMEA) makes the distinction between
constituents with a known therapeutic activity which can be used to standardize a
biological effect and marker compounds which allow standardization on a set
amount of the chosen compound. The EMEA defines marker compounds as
chemically defined constituents of a herbal drug which are of interest for control
purposes, independent of whether they have any therapeutic activity or not.
Examples of markers are the valerenic acids in Valeriana officinalis L.,
ginkgolides and flavonoids in Ginkgo biloba L. and hypericin and hyperforin in
Hypericum perfoliatum.
STABILITY TESTING OF HERBAL PRODUCTS
Stability testing is necessary to ensure the product is of acceptable quality
throughout its entire storage period. An important part of quality control of
herbal products is the evaluation of the chemical stability of a finished product
during the storage period. Stability testing of herbal products is a challenging
task because the entire herb or herbal product is regarded as the active substance,
regardless of whether constituents with defined therapeutic activity are known.
The objective of a stability testing is to provide evidence on how the quality of
the herbal products varies with Stability testing examines the quality and potency
of drug at suitable time intervals under the influence of environmental factors
such as temperature, light, oxygen, moisture, other ingredient or excipient in the
dosage form, particle size of drug, microbial contamination, trace metal
contamination, leaching from the container.
Products are normally required to have shelf lifes that are measured in
years. Accelerated stability studies are designed to increase the rate of chemical
degradation or physical change of a drug substance, therefore, tests must also be
conducted under conditions, which accelerate any changes occurring at ambient
temperature and humidity.

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 With the help of modern analytical techniques like HPLC, HPTLC and by
employing proper guidelines it is possible to establish sound stability data for
herbal products and predict their shelf life which will help in global acceptability
of herbal products.
PHARMACOVIGILANCE OF HERBAL DRUG
Pharmacovigilance is relating to detection, assessment, understanding and
prevention of adverse effects particularly long term and short term effect of
medicines. In other words, it is collecting, monitoring, researching, assessing and
evaluating information from healthcare providers and patients on the adverse
effects of medications. It is also the study of marketed drugs under practical
conditions of clinical usages. Information on adverse drug reactions can be
generated from spontaneous reports or normal clinical trials. Systematic
pharmacovigilance is essential to building up reliable information on the safety
of herbal medicines for the development of appropriate guidelines for safe
effective use. It is to improve patient care and safety in relation to the use of
medicines and all the medical and paramedical interventions, to improve public
health and safety in relation to the use of medicines.
The WHO has welcomed the active participation of drug regulatory
authorities and national pharmacovigilance centers, among others, in the
development of these guidelines. This has provided a useful starting point for
strengthening communication between these authorities, which will be needed to
ensure progress toward the common goal—the safety of herbal medicines. The
guidelines, therefore, identify the particular challenges posed in monitoring the
safety of herbal medicines effectively and propose approaches for overcoming
them. Special attention is also given to the reporting system for adverse reactions
to herbal medicines, and to the analysis of the causes of the reported adverse
reactions.
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 The safety monitoring of herbal medicines is compared and contrasted with
that of other medicines currently undertaken in the context of the WHO
International Drug Monitoring Program. Although there are regulatory and
cultural differences in the preparation and use of different types of medicines,
they are all equally important from a pharmacovigilance perspective. The
inclusion of herbal medicines in pharmacovigilance systems is becoming
increasingly important given the growing use of herbal products and herbal
medicines globally.
Herbal medicines are frequently used in conjunction with other medicines,
and it is essential to understand the consequences of such combined use and
monitor whether any adverse effects are arising. This can be achieved most
readily within the existing pharmacovigilance systems. To handle herbal
medicines and, in particular, to analyze the causes of adverse events, the national
pharmacovigilance centers (or equivalent institutions) will need to acquire
specific technical expertise. This will include trained personnel in the relevant
technical areas and facilities to analyze the products concerned, for which there is
often insufficient information and lack of access to reliable information support.
Many countries currently lack this expertise and, in particular, access to suitable
analytic laboratories. The Member States have therefore recommended the
establishment of regional laboratories specializing in the analysis of herbal
products. The WHO encourages the Member States to explore the feasibility of
this proposal.
REGULATORY STATUS
Phytotherapeutic agents are standardized herbal preparations consisting of
complex mixtures of one or more plants which contain as active ingredients plant
parts or plant material in the crude or processed state. A marked growth in the
worldwide phytotherapeutic market has occurred over the last 15 years. For the
European and USA markets alone, this will reach about $7 billion and $5 billion
per annum, respectively, in 1999, and has thus attracted the interest of most large
pharmaceutical companies. Insufficient data exist for most plants to guarantee
their quality, efficacy, and safety. The idea that herbal drugs are safe and free
from side effects is false. Plants contain hundreds of constituents and some of
them are very toxic, such as the most cytotoxic anti-cancer plant-derived drugs,
digitalis and the pyrrolizidine alkaloids, etc. However, the adverse effects of
phytotherapeutic agents are less frequent compared with synthetic drugs, but
well-controlled clinical trials have now confirmed that such effects really exist.
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Several regulatory models for herbal medicines are currently available including
prescription drugs, over-the-counter substances, traditional medicines and dietary
supplements. Harmonization and improvement in the processes of regulation are
needed, and the general tendency is to perpetuate the German Commission E
experience, which combines scientific studies and traditional knowledge
(monographs). Finally, the trend in the domestication, production, and
biotechnological studies and genetic improvement of medicinal plants, instead of
the use of plants harvested in the wild, will offer great advantages since it will be
possible to obtain uniform and high-quality raw materials which are fundamental
to the efficacy and safety of herbal drugs.
Protocols for standardization of herbal drugs
In order to assure a consistent and acceptable quality herbal product, care
should be taken right from the identification and authentication of herbal raw
materials to the verification process of final product. The following parameters
are recommended.
Recommended protocols
It is desirable to establish a document database containing information on
each approved medicinal herb or herbal medicine. A central digital document
database which is regularly updated and which contains this information with
linkages to references in other databases like NAPRALERT should be
established for easy access by all beneficiaries, producers, and stakeholders. The
knowledge base for an herb or herbal medicine, promoted for wider use, should
be strengthened and expanded so that there is a sound scientific basis for each
use. This would require the presentation of data for each herb, used in herbal
medicine including:
• plant identification, including cultivar with voucher specimen
• plant, preferably voucher stored in a herbarium, for future reference
• age of the herb (maturity/flowering plant, etc.)
• location of cultivation, including altitude and longitude/latitude (GPS)
• fertilizers/pesticides used, if any
• time of harvest, including time after application of pesticides (if
applicable)
• storage conditions of the plant, before sale
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 • drying process
• certificate, confirming the above
Apart from details on each herb, details regarding the manufacture of the
herbal medicine including the following data should be made available:
• protocol or pharmacopoeia or method used for producing the medicine
• plant or plants (for multi-plant herbal medicine) used
• part of plant used in medicine
• vehicle used for producing drug/medicine, e.g., alcohol or water (with
composition if a mixed solvent); type of preservative used, if any, and amount
Where plants are purchased, documents maintained by the supplier regarding the
herb should be made available by the manufacturer.
• Prepare monographs for traditionally used herbal medicines in a suitable
format.
• Legislate to ensure that manufacturers provide relevant data on herbs and
manufacturing processes.
• Initiate programs to conserve biological resources.
• Work toward amending TRIPS to include protection of IPR contained in
indigenous knowledge and to make the development of herbal medicines
attractive to pharmaceutical companies.
• Legislate to establish standards in herbal medicine manufacturing and
ensure their implementation.
• Patents involving biological resources should be granted only if the source
of material is specified and reference made to the material transfer agreement.
Where pharmaceutical companies are prepared to develop herbal medicines into
standardized novel preparations with proven efficacy, the companies could be
provided with IPR, while assuring other stakeholders of a fair share of benefits
on the basis of efficacy or shelf-life being an inventive step. As most herbal
medicines are prepared from more than one plant material, it is imperative that
documentation should be made both on single medicinal plants and on composite
herbal preparations.

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 Single plants
• plant identification: family, genus, species (including cultivar, if any) with
synonyms and older names where applicable; English (common) name(s); local
name
• herbarium voucher; specimen number and date with collector‘s name and
identity
• age of the herb (maturity/ flowering plant, etc.)
• location of cultivation/collection including altitude and longitude/latitude
(GPS)
• fertilizers/pesticides used (if any)
• time of harvest including time after application of pesticides (if
applicable)
• storage conditions of the plant before sale
• drying process
• certificate confirming the above
Composite herbal medicine
Apart from details on each herb as outlined for single plant, details
regarding the manufacture of the composite herbal medicine, including the
following data, should be made available:
• protocol or pharmacopoeia or method used for producing the medicine
• plant or plants (for multi-plant herbal medicine) used
• part of plant or plants used in the medicine
• vehicle used for producing the drug/medicine, e.g., alcohol or water (with
composition if a mixed
• solvent); type of preservative used, if any, and amount
• excipients (if any) used, amount
• major constituents: carbohydrates, protein, fat, dietary fiber, inorganic material,
binder, total energy value per mass (kcal/g or kJ/g)
• suggested dose, number of times to be used, how many days to be used
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• probable side effects/precautions to be taken, contraindications, restrictions for
children, pregnancy, nursing mothers, etc.
• storage: at x °C, room temperature, away from heat, sun, etc.
• stability; shelf life; best before date
• cautionary note, if any
Policies and Regulations
It is a widely held myth that modern drugs are dangerous foreign chemicals
with side effects, while herbals are natural, gentle and safe. The truth is that some
herbs can be dangerous and can bring about serious diseases and even lead to
death. Unlike conventional drugs, herbal products are not regulated for purity and
potency and this could cause adverse effects and can even lead to drug
interactions. There are fewer studies on herbal medicines than on conventional
drugs, mainly because, unlike synthetic chemicals, herbs cannot be patented, so
there is little money to be made by funding such research. It is important that
consumers are made aware of interactions herbs might have with other drugs
they are taking. Unfortunately this information is not available with herbals.
Herbals are also frequently adulterated with prescription drugs. In certain
countries, herbal products used for diagnosis, cure, mitigation, treatment, or
prevention of disease are normally treated as drugs, and hence regulated by
legislation.
However, in most countries, including the United States, such legislation
does not exist and in fact, most botanical products are marketed as dietary supple
ments. Herbal products categorized as nutritional or dietary supplements are not
regulated. In many countries these medicines are not required to pass any
regulatory analysis to be sold as health food supplements.
It is clear that the herbal industry needs to follow strict guidelines and that
regulations are needed. The food and drug administrations that regulate
prescription drugs only review a herbal product if the item is suspected of being
harmful or if the label contains a medical claim. Although research is being done,
it is very limited and only a few herbal drugs have been studied adequately by
well-controlled clinical trials. Even though evidence should always be presented
to support claims of products, most herbs are still marketed with little or no
research. To be registered as drugs, these products need to be tested to prove
their safety and clinical efficacy. However, so far, few programs have been
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established to study the safety and efficacy of herbal medicines as originally
proposed in the WHO guidelines for the assessment of herbal medicines.
The future of herbal drugs is overshadowed by the pervading lack of
regulatory control. In 1993, the WHO sponsored a symposium on the use of
medicinal plants. The result was a standard guideline for the assessment of herbal
medicines and a recommendation that governments of the world should protect
medicinal plants, improve regulation of herbal medicines, and respect traditional
medicine approaches.
More recently the Health Directorate of Canada developed a new regulatory
framework for natural health products, which came into effect in January 2004.
Among other things, the new regulations call for improved labeling, good
manufacturing practices, product and site licensing, and provision of a full range
of health claims that will be supported by evidence. However, even in Canada,
the only regulatory requirements enforced are that all products intended for
medicinal use, including natural health products, are issued a Drug Identification
Number (DIN). These numbers are not required for raw materials such as bulk
herbs.
In the US, access to herbal medicines is restricted by FDA regulations.
Before any new chemical or herbal drug is approved, research must prove that it
is both safe and effective. As a result of these restrictions, packages of herbal
medicines are labeled as food supplements, which do not require pre-approved
testing. Food supplements cannot make any healing claims or issue warnings
about potential risks. In the US, plant-based derivatives already appear in a
quarter of the prescription medicines produced. However, many other plants with
healing properties are shunned by the medical community despite scientific data
from other countries showing their effectiveness. The misconception that herbs
are old fashioned and unscientific has helped to promote a general distrust of
phytotherapy.
Botanical Council contends that, in many cases, herbal medicines are safer
than prescription drugs. According to the Council, herbal medicines react more
slowly and often include their own antidotes to counteract any toxic effects. With
proper enforcement of regulations, more products that are legitimate will enter
the market and the consumers will see justifiable claims on labels. In fact, it is
predicted that appropriate regulations will rejuvenate the market in response to
growing concerns about the regulatory environment for herbal remedies.

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Challenges in Stability testing of herbal medicinal product
Evaluating the stability of HMPs presents a number of challenges when
compared to chemically defined substances. In particular:
1. Active substances (herbal substances and/or herbal preparations) in
HMPs consist of complex mixtures of constituents and in most cases the
constituents responsible for the therapeutic effects are unknown.
2. The situation is further complicated when two or more herbal substances
and/or herbal preparations are combined in a HMP. In many cases where
combinations of herbal substances and/or herbal preparations are present in
HMPs, they have similar constituents and this gives rise to even more analytical
challenges.
3. In addition, many herbal substances/herbal preparations are known to be
unstable.
Taking into account these special features of HMPs, adequate quality
concepts have been established. As part of a total control strategy for herbal
substances, herbal preparations and HMPs, a set of test criteria including
qualitative and quantitative parameters has been recognized as quality indicating.
With regard to stability tests, chromatographic fingerprints as well as appropriate
methods of assay via marker substances represent the fundamental part of this
concept, laid down in shelf-life specifications. Notwithstanding the
appropriateness of this approach, its realization is often associated with analytical
problems and high costs.
In summary, HMPs have a number of characteristics that clearly
differentiate them from chemically defined medicinal products and therefore
specific stability guidance needs to be established, which covers particular
aspects that existing specific herbal guidelines and general guidelines on stability
do not address.
Mechanisms involved in change product
Loss of activity, Change in concentration of active component, Alteration
in bioavaibility, Loss of content uniformity, Loss of elegance, Formation of toxic
degradation product, Loss of packaging integrity.

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Importance of Stability testing:
It evaluates the efficacy of a drug. Stability studies [9] are used to develop
suitable packaging information for quality, strength, purity & integrity of product
during its shelf life. It is used for determination of the shelf life.
Stress testing:
Stress testing help to identify the degradation product, which can help to
establish the degradation pathway. Stress tests are usually considered
unnecessary for herbal drug & its preparation.
1. For herbal drugs and herbal drug preparations, a testing under
accelerated or intermediate conditions may be omitted. This should apply to
finished products as well, because it is known that most products fail at 30°C/65
percent relative humidity (RH) and at 40°C/75 per cent RH in particular. Herbal
drug substances at only 25°C/60 percent RH, with no requirement for
intermediate/ accelerated testing.
2. If intermediate conditions are tested, the three-month time-point is
omitted (that is, 0, 6, 9 and 12 months). In some cases of combination products, it
is hardly possible to provide the required two batches of each extract at the same
time due to different harvesting times.
FINGERPRINTING TECHNIQUES IN HERBAL STANDARDIZATION
INTRODUCTION
Herbal drugs have been used since ancient times as medicines for the
treatment of range of diseases. Medicinal plants have played a key role in the
world health. In recent decades, in spite of the great advances observed in
modern medicine, plants still make an important contribution to health care. This
increased use of herbal medicines is due to several reasons namely, inefficiency
of conventional medicines (e.g. side effects and ineffective therapy), abusive use
of synthetic drugs resulting in side effects, large percentage of world‘s
population does not have access to conventional pharmacological treatment and
folk medicines and ecological awareness which suggest that natural products are
harmless. According to an estimate of the World Health Organization (WHO),
about 80% of the world population still use herbs and other traditional medicines
for their primary health care needs. Of the 252 drugs considered as basic and
essential by the WHO, 11% are exclusively of plant origin and a significant
number of synthetic drugs are obtained from natural precursors. Examples of
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important drugs obtained from plants are digoxin from Digitalis spp., quinine and
quinidine from Cinchona spp., vincristrine and vinblastine from Catharanthus
roseus, atropine from Atropa belladonna and morphine and codeine from Papaver
somniferum. According to WHO, herbal medicines include herbs, herbal
materials, herbal preparations and finished herbal products. Herbs include crude
plant materials, such as leaves, flowers, fruits, seeds, stems, woods, barks, roots,
rhizomes or other plant parts, which may be entire, fragmented or powdered.
Herbal materials in addition to herbs include; fresh juices, gums, fixed oils,
essential oils, resins and dry powders of herbs. Various procedures, such as
steaming, roasting or stir-baking with honey are carried out for their preparation.
Herbal preparations are finished herbal products and may include comminuted or
powdered herbal materials, or extracts, tinctures and fatty oils of herbal
materials. These are produced by extraction, fractionation, purification,
concentration, or other physical or biological processes. Finished herbal products
consist of herbal preparations made from one or more herbs which may contain
excipients in addition to the active ingredients.
STANDARDIZATION
As commercialization of the herbal medicine has happened, assurance of
safety, quality and efficacy of medicinal plants and herbal products has become
an important issue. Herbal raw materials are prone to a lot of variation due to
several factors, the important ones being the identity of the plants and seasonal
variation (which has a bearing on the time of collection), the ecotypic, genotypic
and chemotypic variations, drying and storage conditions and the presence of
xenobiotic. Also one of the major problem faced by herbal drug industry is
unavailability of rigid quality control profile for herbal materials and their
formulations. As per American Herbal Product Association, ―Standardization
refers to the body of information and control necessary to obtain product material
of reasonable consistency. Standardization is a process of evaluating the quality
and purity of herbal drug on the basis of various parameters like morphological,
microscopical, physical, chemical & biological parameters.

166
CONVENTIONAL METHODS FOR STANDARDISATION OF HERBAL
FORMULATION
Phytochemical standardization encompasses all the possible information
generated with regard to the chemical constituents present in an herbal drug.
Hence, the phytochemical evaluation for standardization purpose includes the
following:
 Preliminary testing for the presence of different chemical groups. (e.g., total
alkaloids, total phenolics, total triterpenic acids, total tannins etc.)
 Quantification of chemical groups of interest.
 Establishment of fingerprint profiles based upon single or multiple markers.
Standardization of herbal raw drugs include passport data of raw plant drugs,
botanical authentication which include microscopic & molecular examination,
physical parameters like moisture content, ash value, extractive value etc,
identification of chemical composition by various chromatographic techniques
and determination of biological activity of the whole plant.

167
 Standardization of herbal formulation requires implementation of Good
Manufacturing Practices (GMP). In addition, study of various parameters such as
pharmacodynamics, pharmacokinetics, dosage, stability, self-life, toxicity
evaluation, chemical profiling of the herbal formulations is considered essential.
Good Agricultural Practices (GAP) in herbal drug standardization are equally
important.

ADVANCED TECHNIQUES FOR HERBAL STANDARDIZATION

Quality control of herbal medicines is a tedious and difficult job. Herbal
medicines differ from that of the conventional drugs and so some innovative
methods are necessary for quality assessment of herbal drugs. Fingerprint
analysis approach is the most potent tool for quality control of herbal medicines
because of its accuracy and reliability. Fingerprinting is a process that determines
the concentrations of a set of characteristic chemical substances in an herb.
Knowing the relative concentrations is a means of assuring the quality of herbal
preparations. It can serve as a tool for identification, authentication and quality
168
control of herbal drugs. Based on the conception of phytoequivalence, the
chromatographic fingerprinting and DNA fingerprints of herbal medicines could

be utilized for addressing the problem of quality control of herbal medicines.

Chromatographic fingerprinting:
Chromatographic fingerprinting is the most powerful approach for the
quality control of herbal medicines. Chromatographic fingerprint of Herbal
Medicine is a chromatographic pattern produced from extract of some common
chemical components which may be pharmacologically active or have some
chemical characteristics. This chromatographic profile should be featured by the
fundamental attributions of ―integrity‖ and ―fuzziness‖ or ―sameness‖ and
―differences‖ so as to chemically represent the herbal medicines investigated.
This suggest that chromatographic fingerprint can successfully demonstrate both
―sameness and ―differences between various samples and the authentication
and identification of herbal medicines can be accurately conducted even if the
number and/or concentration of chemically characteristic constituents are not
very similar in different samples of herbal medicine. Thus chromatographic
169
fingerprint should be considered to evaluate the quality of herbal medicines
globally; considering multiple constituents present in the herbal medicines. This
technique can be employed for identification and authentication as well as for
determination of various adulterants and contaminants and for standardization
purpose. In contrast to macroscopic, microscopic and other molecular biological
methods this technique is not restricted to raw herbs, but can also be applied to
pharmaceutical preparations. Chromatographic fingerprinting can be carried out
using techniques such as Thin layer chromatography (TLC), High performance
thin layer chromatography (HPTLC), High performance liquid chromatography
(HPLC), Gas chromatography (GC) and other hyphenated techniques. Thin
Layer Chromatography (TLC) It is one of the most popular and simple
chromatographic technique used of separation of compounds. In the
phytochemical evaluation of herbal drugs, TLC is being employed extensively
for the following reasons:
o It enables rapid analysis of herbal extracts with minimum sample clean-
up requirement.
o It provides qualitative and semi-quantitative information of the resolved
compounds.
In TLC fingerprinting, the data that can be recorded using a high-
performance TLC (HPTLC) scanner which includes information like
chromatogram, retardation factor (Rf) values, the color of the separated bands,

their absorption spectra, λ max and shoulder inflection/s of all the resolved
bands.Eg: TLC fingerprinting was done on the methanolic extract of Sitopaladi
churna for determination of piperine using Silica Gel G plate and Toluene: Ethyl
acetate: Formic acid (5:3.5:0.5 v/v/v) as mobile phase. Retention factor of
piperine was found to be 0.69 (shown by peak 7) at 342 nm.
170
High Performance Thin Layer Chromatography (HPTLC)
HPTLC is the common fingerprinting method mainly used to analyze the
compounds with low or moderate polarities. HPTLC technique is widely used in
the pharmaceutical industry for quality control of herbs and health products,
identification and detection of adulterants, substituents in the herbal products and
also helps in the identification of pesticide contents and Mycotoxins. HPTLC
method has several advantages which are as follows:
o Several samples can be run simultaneously by use of a smaller quantity of
mobile phase as compared to HPLC.
o Mobile phases of pH 8 and above can be used for HPTLC.
o Repeated detection (scanning) of the chromatogram with the same or
different conditions.
o HPTLC has been investigated for simultaneous assay of several
components in a multi-component formulation. With this technique,
authentication of various species of plant as well as the evaluation of stability and
consistency of their preparations is possible.
Eg: HPTLC technique was reported for simultaneous determination of beta-
sitosterol-d glucoside and withaferin A in Ashwagandha formulations using silica

gel GF60 as stationary phase and Chloroform: Methanol (8:2) as mobile phase.
Retention factor was found to be 0.21 (peak 1) and 0.59 (peak 2), respectively.
HPTLC technique is widely employed in pharmaceutical industry in
process development, identification and detection of adulterants in herbal product
and helps in identification of pesticide content, mycotoxins and in quality control
of herbs and health foods (Soni, et al, 2010) It has been well reported that several
samples can be run simultaneously by use of a smaller quantity of mobile phase
171
than in HPLC (Jianga et al., 2010). It has also been reported that mobile phases
of pH 8 and above can be used for HPTLC. Another advantage of HPTLC is the
repeated detection (scanning) of the chromatogram with the same or different
conditions. Consequently, HPTLC has been investigated for simultaneous assay
of several components in a multi-component formulation (Thoppil et al., 2001).
With this technique, authentication of various species of plant possible, as well as
the evaluation of stability and consistency of their preparations from different
manufactures.
Various workers have developed HPTLC method for phytoconstituents in
crude drugs or herbal formulations such as bergenin, catechine and gallic acid in
Bergenia cilliata and Bergenia lingulata (Dhalwal et al., 2008).
High Performance Liquid Chromatography (HPLC)
The preparative and analytical HPLC has been widely used for analysis of
herbal medicines because of its high separation capacity. It can be employed to
analyze almost all constituents of herbal products provided that an optimized
procedure is developed which involves optimization of mobile phase and
stationary phase along with other chromatographic parameters.

172
 There are basically two type of Preparative HPLC Low pressure HPLC
(typically under 5 bar) and High pressure HPLC (pressure >20 bar). Reversed
phase columns are the most popular columns used in the analytical separation of
herbal medicines. In order to obtain better separation , some new techniques are

developed in research field of liquid chromatography like Micellar electrokinetic

capillary chromatography (MECC), High speed counter current chromatography
(HSCCC), Low pressure size exclusion chromatography (SEC), Reversed phase
ion-pairing HPLC (RPIPC-HPLC) and strong anion exchange HPLC (SAX-
HPLC).Eg: Kankasava is a polyherbal formulation prepared with Kanaka and
other ingredients which is used in chronic bronchitis, asthmatic cough and
breathlessness. A simple, precise, accurate RP- HPLC method was developed for
the quantitative estimation of atropine using column RP C-18 (250mm×4.6mm×5
micron) and mobile phase which is mixture of methanol and 10 mM dihydrogen
phosphate buffer in a ratio of 50:50 v/v at a flow rate of 1 ml/min, and analysis
was screened with UV detector at 254 nm. The retention time for standard
atropine sulphate was found to be 4.0667 minutes.
Preparative and analytical HPLC are widely used in pharmaceutical
industry for isolating and purification of herbal compounds. There are basically
two types of preparative HPLC: low pressure HPLC (typically under 5 bar) and
high pressure HPLC (pressure >20 bar) (Chimezie et al., 2008). The important
parameters to be considered are resolution, sensitivity and fast analysis time in
analytical HPLC whereas both the degree of solute purity as well as the amount
of compound that can be produced per unit time i.e. throughput or recovery in
preparative HPLC (Rao et al., 2009). In preparative HPLC (pressure >20 bar),
larger stainless steel columns and packing materials (particle size 10-30 μm are
needed. The examples of normal phase silica columns are Kromasil 10 μm,
Kromasil 16 μm, Chiralcel AS 20 μm whereas for reverse phase are Chromasil
173
C18, Chromasil C8,YMC C18. The aim is to isolate or purify compounds,
whereas in analytical work the goal is to get information about the sample. This
is very important in pharmaceutical industry of today because new products
(Natural, Synthetic) have to be introduced to the market as quickly as possible.
Having available such a powerful purification technique makes it possible to
spend less time on the synthesis conditions (Bhutani, 2000; Marston, 2002;
Brandt et al., 2002).
UNIT-V
BIOLOGICAL TESTS AND ASSAYS OF THE FOLLOWING
Adsorbed Tetanus vaccine:
INTRODUCTION:
• Tetanus vaccine (adsorbed) is a sterile suspension prepared from tetanus
toxoid containing not less than 1000 Limes flocculations (Lf), per mg of
protein nitrogen adsorbed on a mineral carrier, which is hydrated
aluminium hydroxide, aluminium phosphate or calcium phosphate, in saline
soluton or other appropriate solution isotonic with blood.
• The formal toxoid is prepared from the toxin produced by the growth in
suitable media of Clostridium tetani. The toxin is converted to toxoid by
treatment with formaldehyde solution by a method which avoids
reversibility of the toxoid.
Category: Active immunising agent
Biological assay:
The potency of adsorbed tetanus vaccine is determined by comparing the
dose of the vaccine required to protect guinea-pigs or mice from the “paralytic
effects” of a subcutaneous injection of tetanus toxin with the dose of the Standard
Preparation needed to give the same protection.
Standard Preparation:
It consists of a dried mixture of tetanus toxoid adsorbed on aluminium
hydroxide with haemacel (supplied in ampoules containing 340 units) or another
suitable preparation the potency of which has been determined in relation to the
International Standard.
Suggested Method:
Test animal:
Use healthy guinea-pigs from the same stock and weighing between 250-
350g.Distribute the guinea –pigs into six groups of sixteen each. The guinea-pigs
are all of the same sex or the males and females are distributed equally among
the groups. If the challenge toxin to be used has not been shown to be stable or
174
has not been adequately standardized, include four further groups of five guinea-
pigs to serve as unvaccinated controls.
Selection of the challenge toxin:
Select a preparation of tetanus toxin containing not less than 50 times the
50% paralytic dose per ml. If the challenge toxin preparation has been shown to
be stable, it is not necessary to verify the paralytic dose for every assay.
Preparation of the challenge toxin solutions:
Immediately prior to use, dilute the challenge toxin with phosphate
buffered saline pH 7.4 to get a challenge toxin solution containing 50 times the
50% paralytic dose per ml. If necessary, dilute portions of this challenge toxin
solution 16-, 50- and 160-fold with the same buffer solution.
Determination of potency of the vaccine:
• Prepare in saline solution, three dilutions of the vaccine being examined
and three dilutions of a solution of the Standard Preparation such that, for
each, t dilutions form a series differing by not more than 2-5 fold steps and
in which the dilutions of intermediate concentration, when injected
subcutaneously in 1.0ml volumes into guinea-pigs, protect approximately
50% of the animals from the paralytic effects.
• Allocate the six dilutions, one to each of the six groups of sixteen guinea-
pigs and inject subcutaneously 1.0ml of each dilution into each guinea-pig
in the group to which that dilution is allocated.
• After 28 days inject each animal subcutaneously with 1.0ml of the
challenge toxin solution containing 50 times the 50% paralytic dose.
• If necessary, allocate the challenge toxin solution and the three dilutions
made from it one to each of the four groups of five guinea-pigs and inject
subcutaneously 1.0ml of each toxin solution is allocated.
• Examine the guinea-pigs twice daily, remove and kill all animals showing
definite signs of tetanus paralysis. Count the number of guinea-pigs without
paralysis 5 days after injection of the challenge toxin and calculate the
potency of the vaccine being examined relative to the potency of the
Standard Preparation on the basis of the number of animals without
paralysis in each of the six groups of sixteen, using standard statistical
methods.
• For both the vaccines being examined and the Standard Preparation, the
50% protective doses lie between the largest and the smallest doses of the
preparations given to the guinea-pigs.
• The number of paralysed animals among the four groups of five injected
with the challenge toxin solution and its dilutions indicate that the
challenge was approximately 50 times the 50% paralytic dose.

175
 • The limits of the assay fall between 50% and 200% of the estimated
potency.
• The statistical analysis shows no deviation from linearity and parallelism.
Requirement of valid assay:

The test is not valid unless:

i. For both preparations the 50 % protective dose lies between the

largest and smallest dose of preparation given to guinea pigs
ii. The no. of paralyzed animals in 3 groups of 5 injected with the
dilutions of the challenge toxin solution indicate that the challenge
was apprx. 50 times the 50 % paralytic dose.
iii. The reliable limits of assay lie between 50 % and 200 % of the
estimated potency.
Adsorbed Diphtheria vaccine:
 It is a preparation of diphtheria formal toxoid with the mineral
adsorbent.
 Formal toxoid prepared from toxin produced by Coryne bacterium
diphtheria.
 Bulk is prepared by adsorption of bulk purified toxoid onto mineral
carrier – hydrated phosphate or aluminium hydroxide.
Composition:
Each 5ml contains:
Purified diphtheria toxoid – 25 lf
Aluminium phosphate – 2.5 mg
Thiomersal – 0.01%
Category : Active Immunizing agent

176
Identification test :
Solution of 10%w/v of vaccine in sod. Citrate
o
37 C , 16 hrs
centrifuge

clear supernatant liquid

react with diphtheria antitoxin

precipitate is obtained
Absence of toxins:
5 guinea pigs

500 lf purified toxin in 1 ml

None of the guinea pigs shows intoxification with in 6 weeks of injection & 80%
animal survive

if it is – pass if not – fail

Potency:

Three methods are:

 Intradermal challenge method.

 Lethal challenge method.
 Antibody induction method.

177
Principle:
The potency of adsorbed diphtheria vaccine is determined by comparing the
dose of the vaccine required to protect guinea pigs from the effect of either of an
erythrogenic dose of diphtheria toxin administered intradermally or a lethal dose
of diphtheria toxin administered s.c with the dose of a reference preparation.
Intradermal challenge method
The potency of adsorbed diphtheria vaccine is determined by comparing the
dose necessary to protect guinea-pigs against the erythrogenic effects of a range
of intradermal injections of diphtheria toxin with the dose of the Standard
Preparation of adsorbed diphtheria toxoid necessary to give the same protection.
Standard preparation:
The Standard Preparation consist of toxoid adsorbed on aluminium
hydroxide with polygeline (supplied in ampoules containing 132 Units), or
another suitable preparation the potency of which has been determined in relation
to the International Standard.
Suggested method:
1. Selection and distribution of test animals
2. Selection of challenge toxin
3. Preparation of challenge toxin solution
4. Determination of potency of vaccine
5. Determination of activity of challenge toxin
6. Reading and interpretation of results
7. Requirements for a valid assay

Selection and distribution of test animals:

 healthy white guinea pigs from the same stock
 Suitable size for the prescribed number of challenge test
 Difference in body mass between the heaviest and lightest animal being
not greater than 100g
 Divide into 6 equal groups

178
  guinea pigs should be of same sex or with male and female equally
distributed between groups

Selection of challenge toxin:

Diphtheria toxin containing 67 – 133 Lr/100 in 1 Lf and 25000 to 50000
minimal reacting doses for guinea pig skin in 1 Lf.
Preparation of challenge toxin solution:
 Dilute challenge toxin with suitable diluent to obtain a challenge toxin
solution containing about 0.0512 Lf in 0.2 ml
 From this further series of 5 four fold dilutions containing about
0.0128,0.0032,0.0008,0.0002 and 0.00005 Lf in 0.2 ml
Determination of potency of vaccine:
1. Prepare dilutions of the vaccine to be examined and of reference
preparation using 9 g/l solution of sodium chloride.
2. Intermediate dilution when injected s.c at a dose of 1ml/guinea pig –
result an intradermal score of approx. 3
3. After 28 days – inject 0.2 ml of each of the 6 toxin dilutions
intradermally into 6 separate sites on each of the vaccinated pigs.
Determination of activity of challenge toxin:
Inject the unvaccinated control animals with dilutions containing
80,40,20,10,5 millionths of an Lf of the challenge toxin.
Reading and interpretation of results:
• Examine all injection sites 48 hours after injection
• Record the incidence of specific diphtheria erythema
• Record the no. of sites free from such reactions as intradermal
challenge score
Requirement of a valid assay:
The test is not valid unless:
• For both – vaccine to be examined and the reference preparation , the
mean score obtained at the lowest dose level should be less than 3 and
the mean score at the highest dose level should be more than 3
• Toxin dilution that contains 40 millionths of an Lf gives a positive
erythema in at least 80 % of the control guinea groups.
• Toxin dilution containing 20 millionths of an Lf gives a positive
erythema in less than 80 % of the guinea pigs.
179
 • Confidence limits (p=0.95) are not less than 50 % and not more than
200 % of the estimated potency.
• Statistical analysis shows no deviation from linearity and parallelism.
Method of lethal challenge :
i. Selection and distribution of test animals
ii. Selection of challenge toxin
iii. Preparation of challenge toxin solution
iv. Determination of potency of vaccine
v. Determination of activity of challenge vaccine
vi. Reading and interpretation of results
vii. Requirement for a valid assay
Selection of challenge toxin:
Preparation of diphtheria toxin containing not less than 100 LD per ml.
50

Preparation of challenge toxin solution:

Dilute the challenge toxin with suitable diluent to obtain a challenge toxin
solution containing approximately 100 LD Per ml
50

Determination of activity of challenge toxin:

Inject sub cutaneously 1 ml of solution into each guinea pig
Reading and interpretation of results:
Count the number of animal survived 4 days after the injection of the
challenge toxin. Calculate potency using statistical methods.
Requirement for a valid assay:
i.For the vaccine to be examined and the reference preparation the
50% protective dose lies between the largest and smallest dose of
preparation given to guinea pigs
ii. The confidence limit are not less than 50% and not more than 200%
of the estimated potency
iii. The statistical analysis shows no deviation from linearity and
parallelism
Human anti haemophilic vaccine:
Introduction:
 Dried Human Antihaemophilic fraction is a preparation of
Antihaemophilic factor which is obtained from Human Plasma.
 It is rich in clotting factor VIII.
180
Description:-White or pale yellow powder or friable solid.
Storage:-
Stored in atmosphere of nitrogen in light-resistant containers at a temperature
below 8c. The containers are sterile and sealed so as to exclude micro-
organisms.
Production:
 The plasma used is obtained from blood of healthy human donors after
clinical examination, laboratory tests on their blood and consideration of
their medical history i.e free from detectable agents of infection
transmissible by blood transfusion.
 The examinations and tests to be carried out are decided by the
National Regulatory Authority.
In particular, the blood must be tested with negative results for:-
 Evidence of syphilic infection.
 Hepatitis-B surface antigen.
 HIV antibodies by suitably sensitive methods.
 The Haemoglobin value of the donor’s blood is not less than 12.5%w/v.
Assay
Principle:
The potency of human antihaemophilic fraction is determined by comparing
the amount necessary to reduce the clotting time of a test mixture containing
substances that cause clotting of blood with the amount of standard preparation
necessary to produce the same effect under the conditions of the following
method of assay.
Reagents used are:
 Normal serum reagent
 Phospholipid reagent
 Substrate plasma
 Substrate plasma deficient in clotting factor V

181
 Normal serum reagent:
Collect normal human blood in a dry, sterile, glass bottle

shake continuously until coagulation is complete.

Incubate at 37c. for 3 hours, maintain at 4c overnight,

Remove the serum, store at -20c,

Dissolve a quantity of the dried serum in sufficient imidazole buffer pH 7.4 to

produce 10 ml

and allow to stand at 4 c for 16 and 24 hours

Phospholipids reagent:
Suspend 0.125g of Phospholipid in 5ml of water

shake and stir until a uniform suspension is obtained

Prepare a dilution with saline solution that will give the concentration usually lies
between 50 and 250μg per ml

The diluted suspension may be kept at -20c for 6 weeks.

Substrate plasma:
Separate the plasma from 9 volumes of human blood collected in 1
volume of a 3.8%w/v solution of sodium citrate and Store at -20c.
Substrate plasma deficient in clotting factor V:
Separate the plasma from human blood collected in one tenth its volume of
a 1.34%w/v solution of sodium oxalate and incubate at 37c for 24 to 36 hours
and Store in small amounts, at -20c.

182
Preparation of standard and test solution:
Add sufficient imidazole buffer pH 7.4 to produce solutions which contain 0.5 -
2 units per ml
0
these solutions are stable at 20 c for 15min
dilutions are made using a mixture of 1 volume of 3.8%w/v solution of sodium
citrate + 5 volumes of saline solution as diluent.
dilutions should be accurate
Introduce 0.1 of clotting factor V solution + phospholipid reagent + normal
serum reagent into each of six glass incubation tubes

To the first tube add 0.1 ml of the highest dilution of the std preparation, place
the tube in a water-bath at 37c+add 0.1 ml of cacl2 and start stop-watch.

During the next minute add 0.1ml of the second highest dilution of the standard
to a second tube, place it in the water, and add 0.1 ml of 0.05M cacl2 at
exactly 1 minute by the stopwatch.

Repeat the procedure with lowest dilution of the standard and highest to lowest
dilutions of the preparation being examined so that the cacl2 is added at 2, 3, 4
and 5 minutes by the stop-watch, respectively
Place in a water bath at 37c each containing 0.2 ml of 0.025M calcium chloride
and tube containing about 3 ml of substrate plasma.

At 14 minutes transfer 0.1 ml of the mixture from first incubation tube to one of
the tubes containing 0.2ml of 0.025M cacl2 solution and mix

At 15 minutes add 0.2 ml of warmed plasma and record the clotting time
interval between addition of plasma and first indication of fibrin formation
which may be observed visually or by mechanical means.

Repeat the procedure with the other incubation tubes at 1minute interval and
carry out second series of determinations at 21 to 26 minutes.

183
 period of incubation should, be adjusted so that clotting time recorded do not
differ by more than 5%, showing that a stable plateau of prothrombin
activator formation has been reached.

Carry out a blank

Rabies vaccine:
INTRODUCTION:
 Rabies Vaccine, Human (Cell Culture) is a freeze-dried preparation
which is produced on the basis of seed-lot system. It may be prepared by
growing an approved strain of rabies virus in an approved cell culture.
 Animal serum may be used in the medium for initial cell growth but the
medium for maintaining cell cultures during virus multiplication
contains no added protein.
Category: Active immunising agent
STANDARDS
Rabies Vaccine, Human (Cell Culture) has a potency of not less than 2.5
Units per dose
BIOLOGICAL ASSAY OF RABIES VACCINE
The potency of rabies vaccine is determined by comparing the dose
necessary to protect mice against a lethal intra cerebral dose of rabies virus
with the dose of the Standard Preparation of rabies vaccine necessary to
give the same protection
Standard Preparation
The Standard Preparation is the Fifth International Standard, establised
in 1987, containing 16 Units, or another suitable preparation the potency
of which has been determined in relation to the International Standard.
SUGGESTED METHOD:
Test animals: Use mice of a suitable strain,
 Weighing between 11 and 15 g. Distribute the mice into six groups of 16
and four groups of 10
 Mice must be of the same sex or sexes must be equally distributed among
the groups
 Three of the groups of 16 receive the Standard Preparation and the other
three the vaccine being examine
184
 The four groups of 10 are used for titration of the LD 50 of the challenge
suspension
 The strain of mice suitable for the test is such that when 0.03ml containing
5 to 10 LD50 of the standard challenge virus suspension is injected
intracerebrally per mouse there is 100% mortality.
 Throughout the test any mice that die before the fifth day after challenge
are excluded from the test and all mice that die with signs of rabies
between the fifth and fourteenth day after challenge are counted as failing
to resist the challenge.
STANDARD CHALLENGE VIRUS SUSPENSION:
 A working pool of the challenge virus strain is prepared by injecting
intracerebrally 0.03 ml of a 10-fold dilution of the standard strain in 2%
v/v sterile inactivated normal horse serum in water for injection into a
suitable number of test animals.
 The animals when moribund after showing characteristic symptoms of
encephalitis are sacrificed and their brains harvested aseptically. They are
then washed in chilled saline solution to remove blood clots.
 A 10% suspension of the brains is made in 10% acid-digested casein
hydrolysate pH 7.2 and thoroughly homogenised. After centrifuging lightly
the supernatant liquid is distributed into sterile ampoules and freeze-dried.
o o
 The hermetically sealed, freeze-dried ampoules are stored at 2 to 8 . When
stored under the prescribed conditions the virus titre of the freeze-dried
preparation may be expected to be maintained for not less than 3 years.
Alternatively, the washed brains are homogenised in buffered glycerin
solution pH 7.2 to give a 10% suspension.
 It is then centrifuged lightly, distributed into sterile ampoules and
o
hermetically sealed. The sealed ampoules are stored at -20 . In this state,
the virus titre is expected to be retained for not less than 1 year.
DETERMINATION OF POTENCY OF THE VACCINE:
 Reconstitute the Standard Preparation with a suitable liquid. Prepare three
5 serial dilutions of the solution of the Standard Preparation and three 5
serial dilutions of the preparation being examined and allocate one dilution
to each of the six groups of 16 mice.
 Inject intra peritoneally each mouse in each group with 0.5 ml of the
dilution allocated to the group. After 7 days, prepare six similar dilutions
and repeat the injections.
185
 For both the Standard Preparation and the preparation being examined
the serial dilutions should be prepared in such a way that the lowest
dilutions protect from challenge more than 50% of the injected mice
and the highest dilutions protect from challenge less than 50% of the
injected mice.
o
 The hermetically sealed, freeze-dried ampoules are stored at 2 to
o
8 . When stored under the prescribed conditions the virus titre of the
freeze-dried preparation may be expected to be maintained for not
less than 3 years.
 Alternatively, the washed brains are homogenised in buffered
glycerin solution pH 7.2 to give a 10% suspension. It is then
centrifuged lightly, distributed into sterile ampoules and
o
hermetically sealed. The sealed ampoules are stored at -20 . In this
state, the virus titre is expected to be retained for not less than 1
year.
 After a further 7 days inject each vaccinated mouse intracerebrally with
0.03 ml of the standard challenge virus suspension. Observe the mice
for 14 days and count the number of mice surviving the challenge in
each group
 Calculate the potency of the preparation being examined by standard
statistical methods. The test is not valid unless (a) for both the
preparation being examined and the Standard Preparation, the 50%
protective dose lies between the largest and smallest doses given to
the mice; (b) there is no deviation from linearity or parallelism; (c)
the virus titre of the standard challenge virus suspension is between
5 and 50 LD50.

Tetanus Anti toxin:

INTRODUCTION:
• Tetanus means lockjaw, is a medical condition characterised by a
prolonged contraction of skeletal muscle fibres.
• Primary symptoms are caused by tetanospasmin , a neuro toxin
produced by gram +ve, obligate anaerobic bacterium clostridium
tetani.
• Infection generally occurs through wound contamination and
often involves a cut or a deep punture wound.

186
 • As the infection increases, muscle spasm in the jaw develop,
hence the name lock jaw.
• this is followed by difficult in swallowing and generally muscle
stiffness and spasm in other parts of the body.
• It is a preparation containing the specific antitoxic globulines or
their derivatives obtained by purification of hyper immune serum
of horses or other suitable animals
• It has specific activity of neutralizing the toxin formed by
clostridium tetani.
• The liquid preparation may contain a suitable anti microbial
preservative.
• Category: passive immunizing agent
• Dose: by sub cutaneous or intramuscular inj., prophylactic, not
less than 1500 Units; therapeutic, not less than 50,000 Units.
• Freeze dried preparation should be reconstituted in the stated
quantity of diluents supplied by the manufacturer.
• Description: clear, colourless or pale yellow or a freeze dried,
cream coloured powder or pellet.
• Standards: tetanus antitoxin has a potency of not less than
1000Units per ml when intented for prophylactic use and not less
than 3000 Units per ml when intented for therapeutic use.
Biological assay of Tetanus Antitoxin:
Principle:
 The potency of tetanus antitoxin is determined by comparing the
dose necessary to protect mice against the paralytic effects. Fixed
dose of tetanus antitoxin is compared with the dose of the
Standard Preparation of tetanus antitoxin necessary to give the
same protection.
Standard Preparation:
nd
 It is the 2 International Standard consisting of freeze-dried
hyper immune horse serum, the potency is determined in relation
to the International Standard.
Test animals:
 Use healthy mice, weighing between 17 and 22 g, from the same
stock.
Preparation of test toxin:

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  Prepare tetanus toxin from a sterile filtrate of an 8 to 10 days
culture of Cl. tetani.
 Test toxin may be prepared by adding 1 volume of the filtrate to 1
o
or 2 volumes of glycerin, stored below 0 .
 Test toxin prepared in stable form by saturating the filtrate with
ammonium sulphate, collecting the resulting precipitate, drying it
over phosphorus pentoxide and reducing it to a fine powder.
 The powder so obtained is preserved in the dry condition at a
low temperature in sealed ampoules.
Determination of test dose of toxin (Lp/10 dose):
 Limes paralyticum/10 (Lp/10) :This is the smallest quantity of
the toxin mixed with 0.1 Unit of the Standard Preparation and
injected subcutaneously into mice causes paralysis within 4 days.
Procedure:
 Prepare a solution of the Standard Preparation in a suitable liquid
such that 1 ml contains 0.5 Unit.
 Accurately weigh a quantity of the test toxin and dilute it with, a
suitable liquid.
 Prepare mixtures contains 2.0 ml of the solution of the Standard
Preparation (1 Unit), add sufficient of a suitable liquid to give a
final volume of 5.0 ml.
 Allow the mixtures to stand at room temperature, protected from
light, for 60 minutes.
 Inject 0.5 ml of each mixture subcutaneously into mice, six mice
being used for each mixture, observe the mice for 4 days.
 The test (Lp/10) dose of the toxin(0.5 ml) mixture that contains
the smallest amount of toxin sufficient to cause tetanic paralysis
in all six mice injected within 4 days.
Determination of potency of the antitoxin:
Sample preparation for test:
 Prepare a solution of the Standard Preparation in a suitable liquid
such that it contains 0.5 Unit per ml.
 Accurately weigh a quantity of the test toxin and dilute with a
suitable liquid, 1.0 ml contains five times the Lp/10 dose(0.5 Unit
per ml).
Procedure:

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  Prepare mixtures contains 2.0 ml of the solution of the test toxin
+one of a series of graded volumes of the preparation +sufficient
suitable liquid to give a final volume of 5.0 ml.
 Prepare similar mixtures with standard preparation.
 Allow the mixtures to stand at room temperature, protected from
light for 60 minutes.
 Inoculate 0.5 ml of each mixture subcutaneously into each
mouse, six mice being used for each mixture, observe the mice
for 4 days.
 The mixture contains the largest volume of the preparation that
fails to protect the mice from paralysis contains 1 Unit.
 The test is valid only if all the mice injected containing 2.0 ml or
less of Standard Preparation show paralysis and all those injected
with more do not.
 Calculate the potency of the preparation being examined in Units
per ml.
Tetanus Anti serum
Oxytocin:
 Oxytocin is a cyclic nonapeptide hormone obtained by a process of
fractionation from the posterior lobe of the pituitary gland of healthy
oxen or other mammals.
 It has the property of stimulating contraction of the uterus and milk
ejection in receptive animals.
category: oxytocic.
Dose:
 For the induction of labour and for the stimulation of uterine
contractions during labour, by slow intravenous infusion, 1 to 5 Units in
1 litre of 5% Dextrose Injection.
 For control of postpartum haemorrhage, by subcutaneous, intramuscular
or slow intravenous injection, 2 to 5 Units.
Solubility:
 Soluble in water, in 1-butanol and in 2-butanol (for solid).
BIOLOGICAL ASSAY OF OXYTOCIN:
Principle:
 The potency of oxytocin is determined by comparing its activity with that
of the Standard Preparation of oxytocin under the conditions of a suitable
method of assay.

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Method :

 By depression of the blood pressure in chicken — Anaesthetise a young

healthy adult cockerel weighing 1.2 to 2.3 kg with an anaesthetic that will
maintain a prolonged and constant high blood pressure
 Expose the gluteus primus muscle in one thigh and cut and retract it to
reveal the popliteal artery and crural vein. Cannulate the popliteal artery
and record the blood pressure on a suitable recorder calibrated for use over
a linear range. Cannulate the crural or brachial vein.
 Immediately before use prepare a solution of the Standard Preparation in
saline solution so that the volume to be injected is between 0.1 ml and 0.5
ml.
 Record the blood pressure responses to the injection into the cannulated
vein of two doses of this solution.
 The required doses normally lie between 20 and 100 milliUnits. The
interval between injections should be constant and lie between 3 and 10
minutes.
 Immediately before use dilute the preparation being examined with saline
solution so as to obtain responses similar to those obtained with the
Standard Preparation.
 The ratio between the two doses of the preparation being examined should
be the same as that between the two doses of the Standard Preparation and
this ratio should be kept constant throughout the assay.
 The two doses of the Standard Preparation and the two doses of the
preparation being examined should be given according to a randomized
block or a Latin square design and at least six responses to each should be
recorded.
 If the animal rapidly becomes insensitive to the repeated injections of the
solutions another animal must be used. Measure all the responses and
calculate the result of the assay by standard statistical methods.
Heparin sodium IP:
Introduction:
Heparin Sodium is a preparation containing the sodium salt of a complex
organic acid in mammalian tissues and having the characteristic property of
delaying the clotting of shed blood. It is obtained from lungs or intestinal mucosa
of oxen, pigs or sheep and is prepared in conditions to eliminate microbial
conditions
Category: Anti-coagulant

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Standard Preparation and Unit: The Standard Preparation is the freeze-dried
sodium salt of the purified active principle from bovine intestinal mucous
membranes or any other suitable preparation, the potency of which has been
determined in relation to the International Standard.
The Unit is the specific activity contained in 7.7 µg of the Standard Preparation
and is the same as the International Unit; 1 mg contains 130 Units
BIOLOGICAL ASSAY OF HEPARIN SODIUM:
Principle:
The potency of heparin sodium is determined by comparing the concentration
necessary to prevent the clotting with the concentration of the Standard
Preparation of heparin sodium necessary to give the same effect.
Special Reagents:
Prepared Plasma:
 Collect blood from sheep / goats / human volunteers +8% w/v solution of
sodium citrate in the proportion of 1 : 19 volumes of blood to be collected.
 To 1 ml of the pooled plasma add 0.2 ml of a 1% w/v solution of calcium
chloride and mix in a test tube. The plasma is suitable if a solid clot forms
within 5 minutes.
Solution of standard preparation:
 The minimum quantity of the Standard Preparation, when added in 0.8 ml
of saline solution, maintains fluidity in 1 ml of prepared plasma for 1 hour
after the addition of 0.2 ml of a 1% w/v solution of calcium chloride .
Test solution:
 Weigh accurately about 25 mg of the preparation being examined and
dissolve in sufficient saline solution to give a concentration of 1 mg per ml
and dilute to a concentration estimated to correspond to that of the solution
of the Standard Preparation.
Method:
 To test-tubes add graded amounts of the solution of standard preparation
(NMT 0.8 ml).
 To each tube add sufficient saline solution to make the total volume 0.8 ml.
 Add 1.0 ml of prepared plasma to each tube.
 Then add 0.2 ml of a 1% w/v solution of calcium chloride, note the time.
 Repeat as same for test solution, determine the extent of clotting in each
tube, recognizing three grades between zero and full clotting after 1 hr.
 The dilution of the test solution which contains heparin sodium in the same
concentration as the dilution of the solution of standard preparation is that

191
 contained in the series of dilutions which show the same degree of clotting
as the series of dilutions of the solution of standard preparation.
 If the degree of clotting observed in the series of dilutions of the solution
of standard preparation lies between that observed in two of the series of
dilutions of the sample being examined, the potency of the latter is
estimated.
 If there is no such correspondence between the degrees of clotting
produced by the solution of standard preparation and any of the dilutions of
the sample being examined, new dilutions of the latter are prepared and
assay is repeated.
 Not less than 3 independent assays are carried. Calculate the estimated
potency of preparation being estimated by combining the results of these
assays of standard statistical methods
 Limits of error - The limits of error (P = 0.99) attainable with the this test
are:
• 90 and 110%, with three determinations
• 92 and 108%, with four determinations
Antivenom:
QUALITY CONTROL OF ANTIVENOMS
The quality control of the final product is a key element in the quality
assurance of antivenoms. Quality control tests should be performed by the
manufacturer or under its responsibility before the product is released. In
addition, relevant analyses should be performed on any intermediate steps of the
manufacturing protocol as part of the in-process quality control. The results
obtained should meet the specifications approved for each antivenom product or
its intermediates, and are part of the batch record. For a liquid preparation, some
quality control tests, such as the potency test or the detection of residual reagents
used during fractionation, can be performed on the final bulk and may not need
to be repeated on the final product if the processing after the bulk preparation has
been validated and shown not to have any impact. The quality control of the final
product in antivenoms includes the tests described below.
Tests
1.Appearance
The appearance of the product (e.g. colour of the liquid, appearance of the
powder) should comply with the description in the marketing dossier.

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2.Solubility (freeze-dried preparations)
The time from the addition of solvent to the complete dissolution of freeze-
dried antivenom, under gentle mixing, should be determined. Antivenoms should
be completely dissolved within 10 minutes at room temperature. The solution
should not be cloudy. Shaking of the container should be avoided to prevent the
formation of foam.
3.Extractable volume
The volume of product extractable from the container should be in
compliance with that indicated on the label.
4.Venom-neutralizing potency test
This test determines the effectiveness of the antivenom to neutralize the
overall toxic activity of the snake venom(s) against which the antivenom is
designed to act. The first part of the test, to determine the lethal activity of the
venom, is called the median lethal dose (LD50) assay and usually uses mice of a
defined weight range (e.g. 18–20 g). For new venoms whose LD50 is unknown,
it is recommended that a range dose-finding study, using one mouse per venom
dose, is performed to avoid using excessive numbers of animals. Some producers
use other test animals, such as guinea-pigs. While weights will clearly vary
between animal species, the following principles, specified for mice, will still
apply to these alternative test animals. It should be borne in mind that there are
variations in the susceptibility of various strains of mice to the lethal effect of
venoms. LD50 range-finding test: Various venom doses are prepared using saline
solution as diluent, and aliquots of a precise volume (0.2–0.5 ml) of each dose
are injected, using one mouse per dose, by the intravenous route, in the tail vein
(or, alternatively, by the intraperitoneal route (using injection volumes of 0.5
ml)). Deaths are recorded at 24 hours (intravenous test) or at 48 hours
(intraperitoneal test). On the basis of this preliminary dose-finding experiment, a
range of venom doses causing 0% to 100% lethality is established and thus
narrows the range of venom doses required to formally estimate the toxic activity
of the venom.
The median lethal dose (LD50) assay: Groups of 5–6 mice of a defined
weight range are injected intravenously, in the tail vein, with a precise volume
(0.2–0.5 ml) of solutions of varying doses of venom dissolved in sterile saline
solution. A minimum of 5 mice is the smallest number recommended for
obtaining a statistically significant result. In some laboratories the LD50 is
estimated by the intraperitoneal route using an injection volume of 0.5 ml.
Deaths are recorded at 24 hours (for assays involving intravenous injections) or
193
at 48 hours (when intraperitoneal injections are used), and LD50 is estimated by
Probit analysis (102), Spearman-Karber (8) or alternative procedures (such as
non-parametric methods). One venom LD50 is defined as the minimal amount of
venom causing death in 50% of the mice injected. The test to assess the
neutralizing potency of an antivenom is called the median effective dose (ED50)
assay. For a new antivenom, it is recommended that a preliminary range dose-
finding procedure is performed, using one mouse per antivenom dose. ED50
range-finding test: The selected multiple of the venom LD50 (3–6 LD50) is
mixed with different doses of antivenom and incubated at 37oC for 30 minutes
and each mixture
injected into a single mouse. This preliminary test should establish a range of
antivenom volumes that result in 100% survival and 100% death of the injected
mice and thus narrows down the range of doses required for the formal ED(50)
test. The median effective dose (ED50) assay: This test involves the incubation of
a fixed amount of venom (“challenge dose”, usually corresponding to 3–6
LD50), with various volumes of the antivenom adjusted to a constant final
volume with saline solution (53, 103, 104). The mixtures are incubated for 30
minutes at 37 °C, and then aliquots of a precise volume (0.2–0.5 ml) of each
mixture are injected into groups of generally 5 or 61 mice of a defined weight
range by the intravenous route, using the tail vein. A control group injected with
a mixture of the venom “challenge dose” with saline solution alone (no
antivenom) should be included to confirm that the venom “challenge dose”
induces 100% lethality. When the test is performed by the intraperitoneal route, a
volume of 0.5 ml is administered. Centrifugation of the antivenom– venom
mixtures is not recommended because residual venom toxicity may remain in the
immunoprecipitates. After injection, deaths are recorded at 24 hours (intravenous
test) or at 48 hours (intraperitoneal test) and the results analysed using Probit
analysis (102), Spearman- Karber (8) or alternative procedures (such as non-
parametric methods). The median effective dose (ED50) of an antivenom is
defined as the volume of antivenom that protects 50% of the mice injected.
The ED50 can be expressed in various ways:
− mg of venom neutralized by ml of antivenom;
− μl antivenom required to neutralize the “challenge dose” of venom
used;
− μl of antivenom required to neutralize one mg of venom; and
− number of LD50 of venom neutralized per ml of antivenom.

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 Every production laboratory and every national regulatory agency should
establish the accepted levels of neutralizing potency for the various antivenoms
being produced and distributed. In this regard, it is important to guarantee that a
standardized assay is used by the manufacturing laboratories. Since the
methodology to estimate antivenom potency (i.e. ED50) varies between
laboratories and countries, manufacturers should disclose the conditions under
which the potency of their antivenoms is estimated to the corresponding
regulatory agencies in the course of their licensing and control procedures. 1% of
mice may be needed for some venoms.
The protocols for the selection and quality control of the venoms used for
these potency assays should be established in each quality control laboratory (see
section 8). Venoms used in this test should correspond to a representative pool of
well-identified snake specimens collected from various regions within the
geographical range of distribution of the species in a country. These national
reference venom pools must be evaluated periodically to ensure that they have
not deteriorated (see section 8 on quality control of venoms). Until in vitro or
alternative tests of lesser severity become accepted, these venom LD50 and
antivenom ED50 assays should be performed by all manufacturers before an
antivenom can be used in humans. The assays should be conducted under
conditions causing the minimal possible suffering to the experimental animals.
5.Osmolality
Osmolality can be measured to determine the tonicity of the antivenom
solution. It is recommended that it be at least 240 mosmol/kg. Determination of
osmolality is also an indirect means to determine the quantity of salts or
excipients added for formulating the batch.
6.Identification
When several types of antivenoms are produced by a single laboratory, the
identity of each batch of antivenom should be checked. Identity tests may include
biological assays as well as physicochemical and immunological tests. Double
immunodiffusion assays, confronting the antivenom with the venoms against
which the antivenom is designed to act, are often used. In the case of laboratories
that use various animal species to raise antivenoms, i.e. horses and sheep, an
immunological identity test should be used to identify the mammalian species in
which the antivenoms are produced. The potency assay against venoms is another
way to identify antivenoms.

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7. Protein concentration
The total protein concentration of antivenoms is performed using the
Kjeldahl method for nitrogen determination. Alternatively, several colorimetric
procedures can be used, as well as measuring absorbance at 280 nm. The
presence of preservatives should be taken into account since they may interfere
with some protein determination methods .
The total concentration of proteins in antivenoms should preferably not
exceed 10 g/dl, unless a higher protein content is justified and authorized by the
competent authority.
8.Purity
The purity of the active substance, i.e. intact immunoglobulin or
immunoglobulin fragments, should be assessed. They should constitute the great
majority of the preparation, ideally greater than 90%. Electrophoretic methods in
polyacrylamide gels (SDS–PAGE run under reducing or nonreducing conditions)
are suitable for this purpose, since these techniques allow the detection and
monitoring of IgG, F(ab')2, Fab, non-immunoglobulin plasma protein
contaminants (in particular albumin), and degradation products. The
electrophoretic pattern should be compared to that of a reference preparation. A
semi-quantification can be performed by calibration of the procedure.
Of particular relevance is the assessment of the albumin content which
ideally should not exceed 1% of total protein content. The following approach
can serve as a guide in assessing the purity of antivenoms:
− SDS–PAGE under non-reducing conditions. This analysis provides
qualitative (or, at best, semiquantitative) information on the amounts of intact
immunoglobulins, digestion products and, importantly, on the presence of high-
molecular-mass oligomers (soluble aggregates) and low-molecular-mass
contaminants (which are expected in the case of
enzymatically-digested antivenoms).
− SDS–PAGE under reducing conditions. Analysis under these conditions
can provide information on the amount of immunoglobulins and their fragments
by direct visualization of intact and/or digested immunoglobulin heavy chains.
9. Molecular-size distribution
The presence of aggregates and other components in antivenoms can be
assessed by sizeexclusion liquid chromatography (gel filtration) in HPLC
systems. Densitometric analyses of chromatographic profiles allow the
quantification of protein aggregates and of the relative abundances of: intact
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immunoglobulins, divalent immunoglobulin fragments (F(ab')2), monovalent
immunoglobulin fragments (Fab) and dimers, as well as lowmolecular- mass
enzymatic digestion products.
In intact immunoglobulin-based antivenoms this method allows
quantitation of albumin as its molecular mass (~ 66 kDa) can be resolved from
the ~ 160 kDa peak of intact immunoglobulins.
10. Test for pyrogens
Antivenoms should comply with the rabbit pyrogen test where required by
the local regulations. This test is based on intravenous injection of antivenoms in
the ear vein of rabbits (usually 1.0 ml per kg body mass), followed by the
measurement of rectal temperature at various time intervals after injection. The
detailed procedures are described in various pharmacopoeias.
Bacterial lipopolysaccharides can also be detected by the Limulus
amoebocyte lysate (LAL) test. The test should be validated for each type of
antivenom, since there have been reports of falsepositive and false-negative
reactions when testing antivenoms and other plasma-derived products. The
sensitivity of this LAL test should be correlated with the rabbit pyrogen test, and
the endotoxin limits established. When regulation allows, a validated LAL test is
used in place of the rabbit pyrogen test.
11. Test for abnormal toxicity test
The abnormal toxicity test may be performed at the stage of product
development but is increasingly being abandoned in most regulations as it
provides limited information for routine quality assessment of a product. Correct
implementation of GMP should provide evidence that the product would comply
with the test for abnormal toxicity.
12. Test for sterility
Antivenoms should be free of bacteria and fungi, i.e. they should be sterile.
The sterility test is performed following methodologies specified in various
pharmacopoeias such as the European pharmacopoeia. Since antivenoms may
contain preservatives in their formulation, it is necessary to “neutralize” the
preservatives before the samples are added to culture media. This is usually
performed by filtering a volume of antivenom through a 0.45-μm pore
membrane, and then filtering through the same membrane a solution that
neutralizes the bacteriostatic and fungistatic effects of the preservatives used in
antivenom. The membrane is then aseptically removed and cut into two halves.

197
One half is added to trypticase soy broth and the other is added to thioglycolate
medium.
Control culture flasks are included for each medium. Flasks are incubated
at 20–25 ºC (trypticase soy broth) or at 30–35 ºC (thioglycolate) for 14 days.
Culture flasks are examined daily for bacterial or fungal growth. The number of
vials tested per batch should be in compliance with local regulations.
13. Concentration of sodium chloride and other excipients
The concentration of the various excipients or stabilizers added for
formulation should be determined using appropriate chemical methods.
14. Determination of pH
The pH of antivenom should be determined using a potentiometer.
15. Concentration of preservatives
When used in the formulation of antivenoms, the concentration of
preservatives (phenol or cresols) should be quantified. The acceptable range of
preservative concentration in antivenoms should be established and validated in
each quality control laboratory. Phenol concentration should not exceed 2.5 g/l
and cresols 3.5 g/l. Phenol concentration can be determined
spectrophotometrically on the basis of the reactivity of phenol with 4-
aminoantipyrine, under alkaline conditions (pH 9.0-9.2) in the presence of
potassium ferrocyanide as oxidant. Other methods are also available. Cresols can
be determined by HPLC methods.
16. Chemical agents used in plasma fractionation
The chemical reagents used in the precipitation and purification of
antivenoms, such as ammonium sulfate, caprylic acid and others, should be
removed from the final product during diafiltration or dialysis. Limits should be
established and their residual amount quantified in the final product. Likewise,
the elimination of pepsin or papain from the final preparations should be
guaranteed, especially for preparations that are maintained in liquid form, to
avoid proteolytic activity that may damage the antivenoms.
The determination of the residual amount of agents used in plasma
fractionation could be excluded from routine release testing if the process of
manufacturing has been validated to eliminate these reagents. The detection of
residual reagents can also be performed on the final bulk rather than in the final
product.

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17. Residual moisture (freeze-dried preparations)
Residual moisture content can be determined by several methodologies,
such as:
− a gravimetric method assessing the loss of weight on heating;
− the Karl-Fischer titration, based on the principle that iodine, together with
pyridine, sulfur dioxide and methanol from the reagent react quantitatively with
water; and
− thermogravimetric methods.
The methodology most commonly recommended is the Karl-Fischer
titration. Every manufacturing and quality control laboratory must establish the
accepted maximum residual moisture for their antivenom ensuring the stability of
the product over its claimed shelf-life. A residual moisture content of less than
3% is usually recommended for most freeze-dried therapeutic biological
products.
Antivenom reference preparations
The use of in-house reference preparations of antivenoms, instead of
international standards, is recommended, since the potency and specificity can
only be compared with antivenoms of similar specificity and neutralizing profile.
An in-house reference preparation should be obtained from a suitable batch of
the product that has been fully evaluated by the quality control laboratory.
Additional recommended assays for preclinical testing of antivenoms
It is necessary to test whether antivenoms are effective in the neutralization
of the most relevant pathophysiological effects induced by a particular venom.
These “recommended” preclinical tests are, however, not intended for the routine
quality control of antivenom batches. The relevant methods to be used are listed
below.
1. Neutralization of venom haemorrhagic activity
Many venoms, especially those of vipers, exert powerful local and systemic
haemorrhagic activity which is due primarily to venom zinc metalloproteinases.
These enzymes damage the basement membrane that surrounds the endothelial
cells of capillary blood vessels resulting in bleeding into the tissues. Bleeding
into the brain and other major organs is considered to be the major lethal effect of
envenoming by many viperid species (106). The minimum haemorrhagic dose of
a venom (MHD) is defined as the amount of venom (in μg dry weight) which,

199
when injected intradermally, induces in mice a 10-mm haemorrhagic lesion 24
hours after injection.
The MHD test is carried out by preparing aliquots of 50 μl of physiological
saline solution containing a range of venom doses. Mice (18–20 g body weight; 5
mice per group) are placed under light general anaesthesia (e.g.
halothane/oxygen) and the hair surrounding the injection site is shaved. The
venom solutions (50 μl) are injected intradermally in the shaved skin. After 24
hours, mice are killed using an approved humane procedure, the area of the
injected skin is removed, and the haemorrhagic lesion in the inner side of the skin
is measured using calipers in two directions with background illumination. Care
should be taken not to stretch the skin. The mean diameter of the haemorrhagic
lesion is calculated for each venom dose and the MHD estimated by plotting
mean lesion diameter against venom dose and reading off the dose corresponding
to a 10-mm diameter (107, 108).
To estimate the ability of an antivenom to neutralize venom-induced
haemorrhage, a “challenge dose” of venom is selected, which corresponds to one
or more MHDs. Between one and five MHDs have been used as the challenge
dose by different laboratories. The test is carried out as above, using 5 mice per
group. Mixtures of a fixed amount of venom and various dilutions of antivenom
are prepared so that the challenge dose of venom is contained in 50 μl. Controls
must include venom solutions incubated with physiological saline solution alone.
Mixtures are incubated at 37 °C for 30 min, and aliquots of 50 μl are injected
intradermally in lightly anaesthetized mice. The diameter of haemorrhagic
lesions is quantified as described above, and the neutralizing ability of
antivenom, expressed as MHD-median effective dose (ED50), is estimated as the
volume of antivenom, in microlitres, which reduces the diameter of
haemorrhagic lesions by 50% when compared with the diameter of the lesion in
animals injected with the control venom/saline mixture.
2. Neutralization of venom necrotizing activity
Venom-induced local dermonecrosis is a major problem in human victims
of snakebite and it has long been considered important to have an assay system to
evaluate the effect of an antivenom on this pathology. However, it should be
noted that the value of antivenoms in overcoming the cytolytic effects of venoms
has not yet been established; indeed, there is considerable doubt whether
antivenom is useful in obviating such effects in human victims of snakebite. This
is because venom-induced dermonecrosis occurs quickly after a bite and there is
usually a considerable delay between the envenoming of a victim and his or her
arrival in hospital for treatment. Consequently, antivenom therapy can have little
200
or no effect in reversing the damage. Animal experiments in which the
antivenom was administered to the animal at different times after the venom
support this opinion.
The minimum necrotizing dose (MND) of a venom is defined as the least
amount of venom (in μg dry weight) which, when injected intradermally into
groups of five lightly anaesthetized mice (18–20 g body weight), results in a
necrotic lesion of 5 mm diameter 3 days later. The method used is the same as
that for the MHD, except that the skin is examined 3 days after the
intradermal injection of the venom.
To estimate the ability of an antivenom to neutralize venom-induced
dermonecrosis, a challenge dose of venom is selected, usually between one and
two MNDs. The test is carried out as above, using 5 mice per group. Mixtures of
a fixed concentration of venom and various dilutions of antivenom are prepared
so that the venom challenge dose is contained in 50 μl. Controls include venom
solutions incubated with physiological saline solution alone. Mixtures are
incubated at 37 °C for 30 min, and aliquots of 50 μl are injected intradermally in
lightly anaesthetized mice. The diameter of dermonecrotic lesions is quantified 3
days after injection, as described above, and the neutralizing ability of
antivenom, expressed as MND-median effective dose (ED50), is estimated as the
volume of antivenom, in microlitres, which reduces the diameter of necrotic
lesions by 50% when compared with the diameter of the lesion in mice injected
with the control venom/saline mixture.
3. Neutralization of venom procoagulant effect
Many venoms, especially from some vipers, cause consumption of
coagulation factors which results in incoagulable blood. This, combined with the
haemorrhagic nature of some of these venoms, can result in a very poor
prognosis for a severely envenomed patient. Simple in vitro methods exist to
measure this venom-induced pathophysiological effect and the ability of an
antivenom to eliminate it.
The minimum coagulant dose (MCD) of a venom is defined as the least
amount of venom (in mg dry weight per litre of test solution or μg/ml) that clots
either a solution of bovine fibrinogen (2 g/l) in 60 sec at 37 oC (MCD-F) and/or a
standard citrated solution of human plasma (fibrinogen content 2.8 g/l) under the
same conditions (MCD-P).
For measurement of the MCD-F, 50 μl of physiological saline with final
venom concentrations ranging from 240 to 0.5 mg/l is added to 0.2 ml of bovine
fibrinogen solution at 37 oC in new glass clotting tubes. The solutions are mixed
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thoroughly and the clotting time recorded. The MCD-P is estimated by adding
the same venom concentrations to 0.2 ml of the standard human plasma solution
under identical conditions and recording the clotting time. In each case, the MCD
is calculated by plotting clotting time against venom concentration and reading
off the level at the 60-second clotting time.
To estimate the ability of an antivenom to neutralize venom procoagulant
activity, a challenge dose of venom is selected, which corresponds to one MCD-
P or one MCD-F. Mixtures of a fixed concentration of venom and various
dilutions of antivenom are prepared so that the challenge dose of venom is
contained in 50 μl. Controls include venom solutions incubated with
physiological saline solution alone. Mixtures are incubated at 37 °C for 30 min,
and aliquots of 50 μl are added to 0.2 ml of plasma or fibrinogen solution, as
described. The formation or absence of clots is observed during a maximum of
30 min. The minimum volume of antivenom which completely prevents clotting
is estimated and corresponds to the MCD-effective dose.
4. Neutralization of in vivo venom defibrinogenating activity
This test is a direct measure of the in vivo defibrinogenating effect of
certain venoms. To measure the minimum venom defibrinogenating dose
(MDD), a wide range of venom doses is selected and each dose, in a volume of
0.2 ml, is injected intravenously into 4 mice (18–20 g body weight). One hour
after injection, the mice are placed under terminal general anaesthesia and bled
by cardiac puncture. The blood from each animal is placed in a new glass clotting
tube, left at room temperature for 1 hour and the presence/absence of a clot
recorded. The MDD is defined as the minimum dose of venom that produces
incoagulable blood in all mice tested within 1 hour of intravenous injection.
Antivenom neutralization of the venom component(s) responsible for in
vivo defibrinogenation is estimated by incubating a challenge dose of venom,
corresponding to one MDD, with different amounts of the antivenom. Controls
should include venom solutions incubated with saline solution instead of
antivenom. Mixtures are incubated at 37 °C for 30 min before injection of 0.2 ml
by the intravenous route in groups of 4 mice (18–20 g body weight). After 1
hour, mice are bled as described above, the blood is placed in new glass clotting
tubes and left undisturbed for 1 hour at room temperature, after which the
presence or absence of a clot is recorded. Neutralizing ability of antivenoms is
expressed as MDD-effective dose, corresponding to the minimum volume of
antivenom in which the blood samples of all injected mice showed clot
formation.

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5. Neutralization of venom myotoxic activity
The presence of myotoxic components in a venom results in the
degeneration of skeletal muscle by breaking down muscle fibres. Damage is
characterized by the disruption of plasma membranes, local infiltration of
inflammatory cells and oedema. Myotoxicity is characterized by the appearance
of myoglobin in urine and by increments in the serum levels of muscle-derived
enzymes, such as creatine kinase (CK). Myotoxic phospholipase A2 (PLA2)
enzymes are found in a wide range of snake venoms. Some of these PLA2s may
be primarily myotoxic, or neurotoxic, or both. In addition, myotoxicity may
occur as a consequence of ischaemia induced in muscle fibres by the effect of
haemorrhagic venom components in the microvasculature.
Venom myotoxic activity is determined by injecting rats or mice with
various doses of venom in a constant volume of 50 μl (using saline solution as
diluent) into the right gastrocnemius muscle. In the case of mice, groups of 5
animals of 18–20 g body weight are used per dose. Control animals are injected
with the same volume of saline solution. Tail-snip blood samples are collected at
a specific time interval (3 hr in mice), and the CK activity of serum or plasma is
determined using commercially-available diagnostic kits (116, 117). Myotoxic
activity is expressed as the minimum myotoxic dose (MMD), defined as the
amount of venom that induces an increment in serum or plasma CK activity
corresponding to four times the activity in serum or plasma of animals injected
with saline solution alone. Myotoxicity can also be assessed by histological
evaluation of muscle damage after venom injection, although this is a more
expensive and more time consuming method than the CK determination.
To estimate the ability of an antivenom to neutralize venom myotoxicity, a
challenge dose of venom is selected, which corresponds to 3 MMDs. The test is
carried out as above, using 5 mice per group. Mixtures of a fixed concentration
of venom and various dilutions of antivenom are prepared so that the challenge
dose of venom is contained in 50 μl. Controls include venom solutions incubated
with physiological saline solution alone. Mixtures are incubated at 37 °C for 30
min, and aliquots of 50 μl are injected into the gastrocnemius muscle, as
described above.
Blood samples are collected 3 hours after injection (in the case of mice) and
serum or plasma CK activity is quantified. The neutralizing ability of antivenom,
expressed as MMD-median effective dose (ED50) is estimated as the volume of
antivenom, in microlitres, which reduces the serum or plasma CK activity by
50% when compared to the activity of animals injected with venom incubated
with saline solution only (104).
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6. Neutralization of venom neurotoxic activity
Several laboratory methods for assessing venom-induced neurotoxicity
have been developed (e.g. chick biventer cervicis nerve-muscle preparation (118,
119); mouse hemidiaphragm phrenic nerve preparation (120–124), but they are
difficult to perform, require costly equipment and expert technological help and
are unlikely to be practicable for most antivenom producers. Mouse lethality tests
are usually reliable in predicting the neutralization of neurotoxic effects of
venoms.
Development of alternative assays to replace murine lethality testing
In vivo murine assays cause considerable suffering and there have been
calls for the development of alternative assays to replace the standard LD50 and
ED50 tests. The controversy relates to the balance between the clinical benefit to
humans of preclinical testing against the cost to the experimental rodents (death,
pain and distress). This issue is of considerable concern and in vivo tests should
be conducted with the minimal number of animals necessary and using protocols
designed to minimize pain and suffering. There are alternative tests, which
reduce the need for experimental animals, use alternative non-sentient systems or
use in vitro test systems. Unfortunately, such systems cannot currently replace
the rodent toxicity tests.
Consequently, the development of alternative methods to animal testing in
the preclinical evaluation of antivenoms, should be encouraged and when live
animals are absolutely necessary, anaesthesia or analgesia should be considered
and evaluated to ensure that the humane benefits of anaesthesia or analgesia to
the experimental animals do not invalidate the objectives of the assay by altering
relevant physiological processes (53). The establishment of humane end-points to
reduce suffering and limiting the duration of the assays to reduce the period of
animal suffering is also encouraged, but would also need to be carefully
evaluated to ensure the validity of the results.
Limitations of preclinical assays
It is acknowledged that the in vivo and in vitro essential and recommended
preclinical tests have physiological limitations (the venom and venom/antivenom
injection protocols do not represent the natural situation, and the physiological
responses of rodents to envenoming and treatment may differ from those of
humans). Such limitations make the rodent model of human envenoming and
treatment less than ideal. Care should therefore be taken to avoid simplistic
extrapolations from this assay to the clinical situation. Nevertheless, the LD50
and ED50 tests represent the methods most widely used for assessment of
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antivenom potency, and a number of clinical trials have demonstrated that the
ED50 test is useful, but not infallible, at predicting the efficacy of antivenoms in
the clinical setting. An additional value of these tests is the assurance that
antivenoms are manufactured with an accepted, quantifiable and uniform
neutralizing potency.
PCR:
Polymerase chain reaction (PCR) is a technique used in molecular
biology to amplify a single copy or a few copies of a segment of DNA across
several orders of magnitude, generating thousands to millions of copies of a
particular DNA sequence. It is an easy, cheap, and reliable way to repeatedly
replicate a focused segment of DNA, a concept which is applicable to numerous
fields in modern biology and related sciences.
Developed in 1983 by Kary Mullis,PCR is now a common and often
indispensable technique used in clinical and research laboratories for a broad
variety of applications.These include DNA cloning for sequencing, gene cloning
and manipulation, gene mutagenesis; construction of DNA-based phylogenies, or
functional analysis of genes; diagnosis and monitoring of hereditary diseases;
amplification of ancient DNA; analysis of genetic fingerprints for DNA profiling
(for example, in forensic science and parentage testing); and detection of
pathogens in nucleic acid tests for the diagnosis of infectious diseases. In 1993,
Mullis was awarded the Nobel Prize in Chemistry along with Michael Smith for
his work on PCR.

The vast majority of PCR methods rely on thermal cycling, which involves
exposing the reactants to cycles of repeated heating and cooling, permitting
different temperature-dependent reactions—specifically, DNA melting and
enzyme-driven DNA replication—to quickly proceed many times in sequence.
Primers (short DNA fragments) containing sequences complementary to the
target region, along with a DNA polymerase, after which the method is named,
enable selective and repeated amplification. As PCR progresses, the DNA
generated is itself used as a template for replication, setting in motion a chain
reaction in which the original DNA template is exponentially amplified. The
simplicity of the basic principle underlying PCR means it can be extensively
modified to perform a wide array of genetic manipulations. PCR is not generally
considered to be a recombinant DNA method, as it does not involve cutting and
pasting DNA, only amplification of existing sequences.

Almost all PCR applications employ a heat-stable DNA polymerase, such

as Taq polymerase, an enzyme originally isolated from the thermophilic
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bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a
new DNA strand from free nucleotides, the building blocks of DNA, by using
single-stranded DNA as a template and DNA oligonucleotides (the primers
mentioned above) to initiate DNA synthesis.

In the first step, the two strands of the DNA double helix are physically
separated at a high temperature in a process called DNA melting. In the second
step, the temperature is lowered and the two DNA strands become templates for
DNA polymerase to selectively amplify the target DNA. The selectivity of PCR
results from the use of primers that are complementary to the DNA region
targeted for amplification under specific thermal cycling conditions.

Principle of PCR
The PCR involves the primer mediated enzymatic amplification of
DNA. PCR is based on using the ability of DNA polymerase to synthesize new
strand of DNA complementary to the offered template strand. Primer is needed
because DNA polymerase can add a nucleotide only onto a preexisting 3′-OH
group to add the first nucleotide. DNA polymerase then elongate its 3 end by
adding more nucleotides to generate an extended region of double stranded
DNA.

Components of PCR

The PCR reaction requires the following components:

1. DNA Template : The double stranded DNA (dsDNA) of interest, separated

from the sample.
2. DNA Polymerase : Usually a thermostable Taq polymerase that does not
rapidly denature at high temperatures (98°), and can function at a
temperature optimum of about 70°C.
3. Oligonucleotide primers : Short pieces of single stranded DNA (often 20-
30 base pairs) which are complementary to the 3’ ends of the sense and
anti-sense strands of the target sequence.
4. Deoxynucleotide triphosphates : Single units of the bases A, T, G, and C
(dATP, dTTP, dGTP, dCTP) provide the energy for polymerization and the
building blocks for DNA synthesis.
5. Buffer system : Includes magnesium and potassium to provide the optimal
conditions for DNA denaturation and renaturation; also important for
polymerase activity, stability and fidelity.

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Procedure of PCR
All the PCR components are mixed together and are taken through series of 3
major cyclic reactions conducted in an automated, self-contained thermocycler
machine.

1. Denaturation :
This step involves heating the reaction mixture to 94°C for 15-30 seconds.
During this, the double stranded DNA is denatured to single strands due to
breakage in weak hydrogen bonds.
2. Annealing :
The reaction temperature is rapidly lowered to 54-60°C for 20-40 seconds.
This allows the primers to bind (anneal) to their complementary sequence
in the template DNA.
3. Elongation :
Also known at extension, this step usually occurs at 72-80°C (most
commonly 72°C). In this step, the polymerase enzyme sequentially adds
bases to the 3′ each primer, extending the DNA sequence in the 5′ to 3′
direction. Under optimal conditions, DNA polymerase will add about 1,000
bp/minute.

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 With one cycle, a single segment of double-stranded DNA template is
amplified into two separate pieces of double-stranded DNA. These two pieces are
then available for amplification in the next cycle. As the cycles are repeated,
more and more copies are generated and the number of copies of the template is

increased exponentially.

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Types of PCR
In addition to the amplification of a target DNA sequence by the typical PCR
procedures already described, several specialised types of PCR have been
developed for specific applications.

 Real-time PCR
 Quantitative real time PCR (Q-RT PCR)
 Reverse Transcriptase PCR (RT-PCR)
 Multiplex PCR
 Nested PCR
 Long-range PCR
 Single-cell PCR
 Fast-cycling PCR
 Methylation-specific PCR (MSP)
 Hot start PCR
 High-fidelity PCR
 In situ PCR
 Variable Number of Tandem Repeats (VNTR) PCR
 Asymmetric PCR
 Repetitive sequence-based PCR
 Overlap extension PCR
 Assemble PCR
 Intersequence-specific PCR(ISSR)
 Ligation-mediated PCR
 Methylation –specifin PCR
 Miniprimer PCR
 Solid phase PCR
 Touch down PCR, etc

Applications of PCR

Some common applications of PCR in various fields can be explained in

following categories.

Medical Applications:

1. Genetic testing for presence of genetic disease mutations. Eg:

hemoglobinopathies, cystic fibrosis, other inborn errors of metabolism

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 2. Detection of disease causing genes in suspected parents who act as
carriers.
3. Study of alteration to oncogenes may help in customization of therapy
4. Can also be used as part of a sensitive test for tissue typing, vital to organ
transplantation genotyping of embryo
5. Helps to monitor the gene in gene therapy
Infectious disease Applications:
1. Analyzing clinical specimens for the presence of infectious agents,
including HIV, hepatitis, malaria, tuberulosis etc.
2. Detection of new virulent subtypes of organism that is responsible for
epidemics.
Forensic Applications:
Can be used as a tool in genetic fingerprinting. This technology can identify
any one person from millions of others in case of : crime scence, rule out
suspects during police investigation, paternity testing even in case of avaibility of
very small amount of specimens ( stains of blood, semen, hair etc).
Research and Molecular Genetics:
1. In genomic studies: PCR helps to compare the genomes of two
organisms and identify the difference between them.
2. In phylogenetic analysis. Minute quantities of DNA from any source
such a fossilized material, hair, bones, mummified tissues.
3. In study of gene expression analysis, PCR based mutagenesis
4. In Human genome project for aim to complete mapping and
understanding of all genes of human beings.
Selective DNA isolation
PCR allows isolation of DNA fragments from genomic DNA by selective
amplification of a specific region of DNA. This use of PCR augments many
ways, such as generating hybridization probes for Southern or northern
hybridization and DNA cloning, which require larger amounts of DNA,
representing a specific DNA region. PCR supplies these techniques with high
amounts of pure DNA, enabling analysis of DNA samples even from very small
amounts of starting material.

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 Other applications of PCR include DNA sequencing to determine unknown

PCR-amplified sequences in which one of the

amplification primers may be used in Sanger sequencing, isolation of a DNA
sequence to expedite recombinant DNA technologies involving the insertion of a
DNA sequence into a plasmid, phage, or cosmid (depending on size) or the
genetic material of another organism. Bacterial colonies (such as E. coli) can be
rapidly screened by PCR for correct DNA vector constructs. PCR may also be
used for genetic fingerprinting; a forensic technique used to identify a person or
organism by comparing experimental DNAs through different PCR-based
methods.
Some PCR 'fingerprints' methods have high discriminative power and can
be used to identify genetic relationships between individuals, such as parent-child
or between siblings, and are used in paternity testing. This technique may also be
used to determine evolutionary relationships among organisms when certain
molecular clocks are used (i.e., the 16S rRNA and recA genes of
microorganisms).
Electrophoresis of PCR-amplified DNA fragments. (1) Father. (2) Child.
(3) Mother. The child has inherited some, but not all of the fingerprint of each of
its parents, giving it a new, unique fingerprint.
Amplification and quantification of DNA
Because PCR amplifies the regions of DNA that it targets, PCR can be used
to analyze extremely small amounts of sample. This is often critical for forensic
analysis, when only a trace amount of DNA is available as evidence. PCR may
also be used in the analysis of ancient DNA that is tens of thousands of years old.
These PCR-based techniques have been successfully used on animals, such as a
forty-thousand-year-old mammoth, and also on human DNA, in applications
ranging from the analysis of Egyptian mummies to the identification of a Russian
tsar and the body of English king Richard III.
Quantitative PCR methods allow the estimation of the amount of a given
sequence present in a sample—a technique often applied to quantitatively
determine levels of gene expression. Quantitative PCR is an established tool for
DNA quantification that measures the accumulation of DNA product after each
round of PCR amplification.

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 qPCR allows the quantification and detection of a specific DNA sequence
in real time since it measures concentration while the synthesis process is taking
place. There are two methods for simultaneous detection and quantification. The
first method is fluorescent dyes that are retained nonspecifically in between the
double strands. The second method are probes that code for specific sequences
and are fluorescently labeled. Detection of DNA using these methods can only be
seen after the hybridization of probes with its complementary DNA takes place.
An interesting technique combination is real-time PCR and reverse transcription
(RT-qPCR). This sophisticated technique allows for the quantification of a small
quantity of RNA. Through this combined technique, mRNA is converted to
cDNA, which is further quantified using qPCR. This technique lowers the
possibility of error at the end point of PCR, increasing chances for detection of
genes associated with genetic diseases such as cancer. Laboratories use RT-
qPCR for the purpose of measuring gene regulation sensitively.
Gene Expression Profiling and Quantitation: Methods and Techniques
Researchers studying gene expression employ a wide variety of molecular
biology techniques and experimental methods. Gene expression analysis studies
can be broadly divided into four areas: RNA expression, promoter analysis,
protein expression, and post-translational modification.
RNA Expression

 Northern blotting — steady-state levels of mRNA are directly quantitated

by electrophoresis and transfer to a membrane followed by incubation with
specific probes. The RNA-probe complexes can be detected using a variety
of different chemistries or radionuclide labeling. This relatively laborious
technique was the first tool used to measure RNA levels
 DNA microarrays — an array of oligonucleotide probes bound to a chip
surface enables gene expression profiling of many genes in response to a
condition. Labeled cDNA from a sample is hybridized to complementary
probe sequences on the chip, and strongly associated complexes are
identified optically. Gene expression profiling is often a first step in a gene
expression analysis workflow, investigating changes in the expression
profile of a whole system or examining the effects of mutations in
biological systems
 Real-Time PCR — steady-state levels of mRNA are quantitated by reverse
transcription of the RNA to cDNA followed by quantitative PCR (qPCR)
on the cDNA. The amount of each specific target is determined by
measuring the increase in fluorescence signal from DNA-binding dyes or
probes during successive rounds of enzyme-mediated amplification. This
precise, versatile tool is used to investigate mutations (including insertions,
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 deletions, and single-nucleotide polymorphisms (SNPs)), identify DNA
modifications (such as methylation), confirm results from northern blotting
or microarrays, and conduct gene expression profiling. Expression levels
can be measured relative to other genes (relative quantification) or against a
standard (absolute quantification). Real-time PCR is the gold standard in
nucleic acid quantification because of its accuracy and sensitivity. Real-
time PCR can be used to quantitate mRNA or miRNA expression following
conversion to cDNA or to quantitate genomic DNA directly to investigate
transcriptional activity

Quantitative PCR instrument

A quantitative PCR instrument is a machine that amplifies and detects
DNA. It combines the functions of a thermal cycler and a fluorimeter, enabling
the process of quantitative PCR.

The first quantitative PCR machine was described in 1993, and two
commercial models became available in 1996. By 2009, eighteen different
models were offered by seven different manufacturers. Prices range from about
20,000 USD to 150,000 USD

Principal performance dimensions of quantitative PCR instruments are

thermal control, fluorimetry and sample throughput.

Thermal control

Efficient performance of quantitative PCR requires rapid, precise, thermal

control.

30 cycles of PCR have been demonstrated in less than 10 minutes. Rapid

cycling provides several benefits, including, reduced time to result, increased
system throughput and improved reaction specificity. In practice however,
engineering trade-offs between ease of use, temperature uniformity, and speed,
mean that reaction times are typically more than 25 minutes.

Thermal non-uniformity during temperature cycling contributes to

variability in PCR and, unfortunately, some thermocyclers do not meet the
specifications claimed by manufacturers.[10] Increasing the speed of thermal
cycling generally reduces thermal uniformity, and can reduce the precision of
quantitative PCR.

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 The temperature uniformity also has a direct effect on the ability to
discriminate different PCR products by performing melting point analysis. In
addition to uniformity, the resolution with which instruments are able to control
temperature is a factor which affects their performance when performing high
resolution melting analyses.

Therefore speed, precision and uniformity of thermal control are important

performance characteristics of quantitative PCR instruments.

Fluorimetry

Quantitative PCR instruments monitor the progress of PCR, and the nature
of amplified products, by measuring fluorescence.

The range of different fluorescent labels that can be monitored, the

precision with which they can be measured, and the ability to discriminate
signals from different labels, are relevant performance characteristics.

By using an instrument with sufficient optical channels and extensive assay

optimisation, up to 7 separate targets can be simultaneously quantified in a single
PCR reaction. However, even with extensive optimisation, the effective dynamic
range of such multiplex assays is often reduced due to interference between the
constituent reactions.

The noise in fluorescence measurements affects the precision of qPCR. It is

typically a function of excitation source intensity variation, detector noise and
mechanical noise. Multi factorial analysis has suggested that the contribution of
mechanical noise is the most important factor, and that systems with no moving
parts in their optical paths are likely to provide improved quantitative precision.

In addition, when performing high resolution melting analyses, one factor

that affects the sensitivity of heteroduplex detection is fluorimetric precision.

Therefore the number of optical channels and the level of noise in

fluorescence measurements are also important performance characteristics of
quantitative PCR instruments.

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 UNIT-VI
IMMUNOASSAY:
Immunoassays are chemical tests used to detect or quantify a specific
substance, the analyte, in a blood or body fluid sample, using
an immunological reaction. Immunoassays are highly sensitive and specific.
Their high specificity results from the use of antibodies and purified antigens as
reagents. An antibody is a protein (immunoglobulin) produced by B-lymphocytes
(immune cells) in response to stimulation by an antigen. Immunoassays measure
the formation of antibody-antigen complexes and detect them via an indicator
reaction. High sensitivity is achieved by using an indicator system
(e.g., enzyme label) that results in amplification of the measured product.
Immunoassays may be qualitative (positive or negative) or quantitative
(amount measured). An example of a qualitative assay is an immunoassay test for
pregnancy. Pregnancy tests detect the presence of human chorionic gonadotropin
(hCG) in urine or serum. Highly purified antibodies can detect pregnancy within
two days of fertilization. Quantitative immunoassays are performed by
measuring the signal produced by the indicator reaction. This same test for
pregnancy can be made into a quantitative assay of hCG by measuring the
concentration of product formed.
The immunoassay is a technique which incorporates the binding reaction of
a target substance (antigen) with an antibody. Antibodies are basically
immunoglobins that bind to different natural and synthetic antigens in the body
such as carbohydrates, lipids, proteins and nucleic acids. The antibodies have a
common structure but have different special components that help them identify
and bind to specific antigens. This results in the flexibility needed for effective
immunity in a heterogenic environment. The specificity of antibodies which
enable them to bind to different antigens is shown in Figure 2.1 below:

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 Immunogens, also known as antigens are substances commonly proteins
which are coupled to hapten (a carrier) that can cause the formation of antibodies
when it is introduced in a host body. The mechanism exhibited by antigen and
antibody is like a ‘lock and key’ one which determines the affinity and binding
pattern of these substances in a solution. The core concept of immunoassay is the
‘antigen-antibody’ binding reaction which has a great impact on assay
effectiveness and development. These reaction are however, quite variable.

In a normal host, after immunization, poly colonal anti sera is generated.

This anti sera is a heterogeneous mixture of antibodies which have different
binding specificities and affinities. The assays which have polyclonal antibodies
would have a broader range of cross reactivity, which depends on their
application. It is quite easy and cheap to produce polyclonal antisera.

Isolated cloned cells produce mono colonal antibodies. In 1975, George

Kohler and Cesar Milstein produced hybrid cells which were formed by the
fusing of B lymphocytes and immortal myeloma cells. These hybrid cells, also
known as hybridomas, were able to generate very specific antibodies which can
differentiate between a variety of target molecules. These antibodies were
invaluable to the field of therapeutics and diagnosing. When compared to poly
colonal antibodies, the mono colonal antibodies were much more specific for
immunoassays and provided continuous antibodies. Recombination of DNA
technology is now used to further improve the flexibility and specificity of this
technology.

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Purpose
The purpose of an immunoassay is to measure (or, in a qualitative assay, to
detect) an analyte. Immunoassay is the method of choice for measuring analytes
normally present at very low concentrations that cannot be determined accurately
by other less expensive tests. Common uses include measurement of drugs,
hormones, specific proteins, tumor markers, and markers of cardiac injury.
Qualitative immunoassays are often used to detect antigens on infectious agents
and antibodies that the body produces to fight them. For example, immunoassays
are used to detect antigens on Hemophilus, Cryptococcus ,
and Streptococcus organisms in the cerebrospinal fluid (CSF) of meningitis
patients. They are also used to detect antigens associated with organisms that are
difficult to culture, such as hepatitis B virus and Chlamydia trichomatis .
Immunoassays for antibodies produced in viral hepatitis, HIV, and Lyme disease
are commonly used to identify patients with these diseases.
Principles Regarding The Methodology Of Immunoassays
Immunoassays have been classified as being:

 Competitive and Non competitive

 Heterogeneous and Homogenous
The immunoassays basically identify a label which is detected to measure the
amount of antigen or antibody which is present in a sample. These ‘labels’ can
either be radioactive isotopes or enzymes that cause changes in color or produce
light. Depending on the type of assays, labels either exist on the antigen or the
antigen.

Limited reagent or competitive immunoassays are related to the phenomenon

of the sample’s analyte and the labeled analyte’s competition for the antibody.
(See Figure 2.2) The signal’s measurement would indicate the amount of target
compound that is present. Mostly, primary assays types are the competitive
assays that are being utilized in the drug testing programs of workplaces.

While doing the noncompetitive immunoassays, the antibody is adsorbed on

the solid phase’s surface and the sample interacts with the solid phase. This
system has excessive labeled antibodies in it which bind to the whole target
analyte. Then, another labeled antibody is inserted which results in the
sandwiching of the target analyte. The sample is then measured to know the
quantity of the analyte which is there in the sample. That is why, non competitive
assays are also known as ‘2-site’ or ‘sandwich’ immunoassays.

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 Heterogeneous assays are those which require the separation of bound and free
labels. In heterogeneous assays, the portion of antigen bound with the antibody is
disconnected from the remaining unbound part after the reaction takes place.
This separation of assays is done via solid phase adsorption, liquid phase
adsorption or precipitation.

However, homogenous assays are those where the label is changed by the
binding process, thereby allowing the binding to be observed without separation
step. These systems are marked by their speed and ease and can be easily
automated. These assays are commonly used to monitor therapeutic drug
measurement and also, illicit drug testing programs for workplaces where low
detection limits are not needed.

REAGENTS REQUIRED FOR IMMUNOASSAY DEVELOPMENT

These reagents are the antibodies, signal-generating labels, and separation
matrices. Antibodies are the key reagents on which the success of any
immunoassay depends. The antibodies can be either polyclonal or monoclonal.
However, for immunoassay development for pharmaceutical analysis purposes,
monoclonal antibodies are more advantageous than polyclonal ones. This is
attributed to their higher degree of affinity and specificity towards the analyte.
Even that, many successful immunoassays were developed using polyclonal
antibodies because it was possible to generate the antibodies with high affinity to
the analyte.

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 The signal generating labels in immunoassays include radioactive atoms
(mostly 125I, 3H, and 14C). The use of radioactive labels offers extremely
sensitive and quite precise assays, however, they have some drawbacks (e.g.
health hazards, special attention for handling of the reagents, training of staff,
short half-life time of the isotope, and expensive instrumentation for the counting
of radioactivity. Therefore, alternative non-radioactive labels such as enzyme,
fluorescent probes , chemiluminescent substances, metals and metal chelates, and
liposomes were introduced. On the basis of number of publications, enzymes are
the most common labels employed in immunoassay methods for pharmaceutical
compounds. A potential advantage in the use of enzyme labels for immunoassay
is the possibility of the amplification of the signal, and subsequently the potential
increasing in the sensitivity of the method. This is beneficial when the original
signal is not sufficient to get the desirable sensitivity for the analysis.
The matrices used for separation of the immune complexes that formed as a
result of immunoanalytical reactions include charcoal, polyethylene glycol,
second antibody, microbeads or the most useful 96-well microwell plates; each
well of the plate serves as a separate reaction tube. One component of the
reaction (analyte or antibody) is coated onto the surface of the bottom of the plate
wells, and the immune complex is formed on the surface of the wells. The use of
these plates facilitates the washing steps, and reagents pipetting, and thus leads to
semi-automation of the method.

BASIC METHODOLOGY INVOLVED IN PHARMACEUTICAL

ANALYSIS
Immunoassay methods that have been applied in pharmaceutical analysis,
based on whether the separation step is or is not required, can be classified into
heterogeneous or homogeneous assay, respectively. These methods can be
performed in either competitive or non-competitive designs. The choice from
these designs is based on nature of the analyte, labeling chemistry available and
the analytical parameter required from the assay (e.g. sensitivity, dynamic range,
and precision). Competitive design of immunoassays can be carried out in an
antigen-capture or antibody-capture format, depending on whether the solid
phase is coated with antibody or antigen (analyte), respectively. The features of
these formats are shown in Figure1.1. In the antigen-capture format (Figure 1A),
the competition reaction occurs between the analyte (in sample) and a labelled
analyte for the binding to a limited amount of anti-analyte antibody coated onto a
solid support. After equilibration and separation, the label activity on the solid
phase is measured, and the measured signal is inversely correlated to the
concentrations of analyte in the sample. In antibody-capture format (Figure 1B),
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the analyte (or its protein conjugate) is coated onto a solid support. The
competition occurs between the analyte (in sample) and the immobilized analyte
for the binding to a limited amount of labelled anti-analyte antibody. After
equilibration and separation, the activity of the label bound to the solid support is
measured, and the signal is inversely correlated to the concentration of the
analyte. The non-competitive design (usually called “two-site” or “sandwich”
assay) is used for large analytes possessing more than one recognition epitopes
on the molecule. It requires two antibodies that bind to non-overlapping epitopes
on the analyte molecules. One of the two antibodies is bound to the solid phase,
and the second one is labelled and used for detection. Figure 2 illustrates the
features of this assay. The sample analyte is allowed to bind to an immobilized
antibody. After washing, the solid support (contains the formed analyte-antibody
complex) is incubated with an excess of the second labelled antibody, which
binds to the remaining epitope on the analyte molecule. After washing, the
activity of the label bound to the solid support is measured.

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221
222
Production of Antibodies

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 The production of antibodies is an important process in the use of
immunoassays because it is the antibody-antigen complexes that form that the
device uses for its results. Antibodies can be called monoclonal or polyclonal,
depending upon the technique used to produce it. Monoclonal antibodies involve
these basic steps and result in very specific antibodies that bind only to one
antigen epitope, which in turn reduces the occurrence of false positives in the
immunoassay:
− A mouse (or rabbit) is immunized by being injected with an antigen; the
antigen generates an antibody response in the animal. − The mouse is sacrificed
and the antibody forming cells are isolated from the mouse's spleen.
− Monoclonal antibodies are produced by fusing single antibody-forming
cells with myeloma cells. The resulting cell is called a hybridoma. This
hybridization makes the cells “immortal.”
− The hybridomas contain large amounts of antibodies, and can easily be
cultured into populations of cells that contain identical antibodies. These
antibodies are called "monoclonal antibodies" because they are produced by the
identical offspring of a single, cloned antibody producing cell.

224
A diagram depicting the steps involved in monoclonal antibody production
Polyconal antibodies are more likely to produce a false positive because
they are less specific to antigen epitopes and have varying binding affinities; they
may bind of molecules similar to their antigens. Production includes these steps:
− A mouse (or rabbit) is immunized by being injected with an antigen; the
antigen generates an antibody response in the animal.
− The animal is bled and the antibodies are collected; the blood contains a
heterogenous mixture of antibodies of varying binding affinities and specificities.
Immunoassay Results
• Qualitative – Single point calibration at a specific cutoff. Results are
either ‘positive’ or ‘negative’; (i.e. above or below the cutoff). Possible false
positives; monoclonal antibodies restrict this slightly.
• Quantitative – Provides numeric results that are an estimate of
drug/compound concentration based on the measurement of labeled analyte in
the solution, and taking into consideration the competitive/ noncompetitive
nature of the device. In terms of use on drugs, this is sometimes complicated by
possible cross-reactivities.

225
Typical 4-parameter logistic graph for a competitive-format immunoassay.

Dose-response curve for a noncompetitive CL immunoassay.

A
pregnancy test is an example of a commercially produced immunoassay that produces a
positive or negative qualitative response.
Standardization of Immunoassays
The aim of standardization is to ensure that assays of the same analyte in
the same samples, done at different places or at different times or both, can be
readily compared. International Standards are prepared by the National Institute
for Biological Standards and Control in the UK. Preparation of standard proteins
are purified, mixed with an inert carrier compound, divided and freezedried, and
sent to laboratories. Known standards include: – Cortisol – Alphafetoprotein

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(AFP) – Carcino-embryonic antigen (CEA) – Human gonadotropin (hCG)

Cortisol

Preparation of Polyclonal and Monoclonal Antibodies

Antibody reagents are developed from either polyclonal or monoclonal
antibodies. Polyclonal antiserum (serum from blood containing the desired
antibodies) is generated in animals, most commonly sheep, rabbits, or goats. The
animals produce the antiserum - just as a human would - as a defense mechanism
when exposed to an antigen. Antiserum contains a mixture of antibodies, each of
which may bind to different antigen binding sites, or epitopes.
Producing antiserum
The process of making an antiserum begins by injecting a solution that
contains the antigen of interest into an animal. This antigen of interest is
sometimes called an immunogen, because it can stimulate an immune response.
Over time, and in some cases with multiple injections, the immune system of the
animal produces antibodies to the antigen that was injected. Blood is collected
from the animal, and serum is isolated from the blood. This serum is usually rich
in antibodies that recognize the antigen, and is called the antiserum.
Polyclonal antibodies
Antiserum usually contains a mixture of antibodies that recognize and bind to
the same antigen, but they may attach to different epitopes

227
 (see
Figure 1-2). An antigen that has multiple sites for antibodies to bind is called a
multivalent antigen. These types of antibodies, present as a diverse mixture, are
called polyclonal antibodies.
Monoclonal antibodies
Monoclonal antibodies differ from polyclonal antibodies in that they are
highly specific for a single epitope on a multivalent antigen

(see
Figure 1-3). They are produced from a single cell line using hybridoma
technology and mouse myeloma cell lines. Hybridomas are antibody-producing
tumor cells that produce many copies of the same antibody and grow easily in
laboratory cell culture.

228
An advantage
An advantage of monoclonal antibodies is that the hybridoma cell line that
produces them is potentially “immortal” and can produce the same antibodies
consistently and indefinitely. A polyclonal antisera produced by immunization of
animals can vary from animal to animal, and a useful antiserum may no longer be
available if the single animal that produces it dies procedure Hybridomas are
produced in a multi-step procedure

229
230
(see Figure 1-4):
• Injecting a specific antigen into a host animal (typically a mouse);
• Isolating antibody-producing cells (B cells) from the spleen of the mouse;
• Fusing these B cells with a specific type of tumor cell that grows easily in
culture and produces antibodies;
•Isolating successful hybridomas (fused cells) that produce antibodies specific
for the antigen of interest.
• In immunoassays, both monoclonal and polyclonal antibodies are used for
detecting antigens, each with specific strengths for particular applications.
• Immunoassays that detect antibodies in patient sera are likely to involve
detection of polyclonal antibodies generated by the patient’s immune system.
Radioimmunoassay
INTRODUCTION
To perform a radioimmunoassay, a known quantity of an antigen ismade
radioactive, frequently by labeling it with gamma-radioactive isotopes of iodine
attached to tyrosine. This radiolabeled antigen is then mixed with a known
amount of antibody for that antigen, and as a result, the two specifically bind to
oneanother.Then, a sample of serum from a patient containing an unknown
quantity of that same antigen is added. This causes the unlabeled (or "cold")
antigen from the serum to compete with the radiolabeled antigen ("hot") for
antibody binding sites. As the concentration of "cold" antigen is increased, more
of it binds to the antibody, displacing the radiolabeled variant, and reducing the
ratio ofantibody-bound radiolabeled antigen to free radiolabeled antigen. The
bound antigens are then separated from the unbound ones, and theradioactivity of
the free antigen remaining in the supernatant is measuredusing a gamma counter.
The RAST test (radioallergosorbent test) is an example ofradioimmunoassay.
It is used to detect the causative allergen foran allergy
Radioimmunoassay (RIA) is a very sensitive in vitro assay techniqueused to
measure concentrations of antigens (for example, hormone levels intheblood) by
use of antibodies.
AgAb + Ag*Ab + Ag + Ab*
◦ Unbound Ag* and Ag washed out
◦ Radioactivity of bound residue measured
◦ Ligand conc is inversely related to radioactivity [Ag : ligand to be measured
; Ag* radiolabelled ligand]
Principle:
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 Uses an immune reaction [Antigen – Antibody reaction] to estimate a ligand
Ag + Ag* + Ab
Advantages
◦ Highly specific: Immune reactions are specific
◦ High sensitivity : Immune reactions are sensitive
Disadvantages of RIA
o Radiation hazards: Uses radiolabelled reagents
o Requires specially trained persons
o Labs require special license to handle radioactive material
o Requisition, storage of radioactive material
o Requires special arrangements for radioactive waste disposal
Requirements for RIA:
1. Preparation & characterisation of the Antigen [Ligand to be analysed]
2. Radiolabelling of the Antigen
3. Preparation of the Specific Antibody
4. Development of Assay System
Preparation & Radiolabelling [Tagging procedure]
◦ 3 H 14 C 125 I are used as radioactive tags

◦ Antigens are tagged to 3 H 14 C 125

◦ Tagging should NOT affect Antigenic specificity
Antigens prepared by..
◦Synthesis of the molecule
◦Isolation from natural sources
◦Radiolabelling of the Antigen & Antigenic activity

Radioimmunoassay (RIA)
The greater the specificity of the antiserum, the greater the specificity of the
assay. Using antibodies of high affinity, it is possible to detect a few picograms
(10−12 g) of hormone in the tube. Radioimmunoassay is widely-used because of
its great sensitivity.
The body concentrates iodine atoms — radioactive or not — in the thyroid
gland where they are incorporated in thyroxine (T4). Both 125I or 131I emit
gamma radiation that requires special counting equipment. The main drawbacks
to radioimmunoassay are the expense and hazards of preparing and handling the
radioactive antigen.

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INSTRUMENTATION
Radiation will hit silver grains in emulsion and expose them to film
or emulsion. Isotope will emit radiation (usually beta) Incubate tissue with
radioactive ligand .

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Method
Classically, to perform a radioimmunoassay, a known quantity of
an antigen is made radioactive, frequently by labeling it with gamma-
radioactive isotopes of iodine, such as 125-I, attached to tyrosine. This
radiolabeled antigen is then mixed with a known amount of antibody for that
antigen, and as a result, the two specifically bind to one another. Then, a sample
of serum from a patient containing an unknown quantity of that same antigen is
added. This causes the unlabeled (or "cold") antigen from the serum to compete
with the radiolabeled antigen ("hot") for antibody binding sites. As
the concentration of "cold" antigen is increased, more of it binds to the antibody,
displacing the radiolabeled variant, and reducing the ratio of antibody-bound
radiolabeled antigen to free radiolabeled antigen. The bound antigens are then
separated from the unbound ones, and the radioactivity of the free(unbound)
antigen remaining in the supernatant is measured using a gamma counter.
This method can be used for any biological molecule in principle and is not
restricted to serum antigens, nor is it required to use the indirect method of
measuring the free antigen instead of directly measuring the captured antigen.
For example, if it is undesirable or not possible to radiolabel the antigen or target
molecule of interest,a RIA can be done if two different antibodies that recognize
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the target are available and the target is large enough (e.g., a protein) to present
multiple epitopes to the antibodies. One antibody would be radiolabeled as above
while the other would remain unmodified. The RIA would begin with the "cold"
unlabeled antibody being allowed to interact and bind to the target molecule in
solution. Preferably, this unlabeled antibody is immobilized in some way, such as
coupled to an agarose bead, coated to a surface, etc. Next, the "hot" radiolabeled
antibody is allowed to interact with the first antibody-target molecule complex.
After extensive washing, the direct amount of radioactive antibody bound is
measured and the amount of target molecule quantified by comparing it to a
reference amount assayed at the same time. This method is similar in principle to
the non-radioactive sandwich ELISA method.
Radioimmunoassay can detect substance like :
• Hormones
• Vitamins
• Serum Protein
• Drugs
• Infective Agent
Sensitivity of Radioimmunoassay :
It can detect substance from a range of Nano gram(ng) to Pico gram(pg)
concentration. Qualitative as well as Quantitative analysis. Advantages of
Radioimmunoassay
• Specific
• Sensitive
• Convenient
• Reliable
• Reproducible
Application Of Radioimmunoassay
1)Detection of Narcotic Drugs Heroin & Morphine can be detected in hair
with the use of Radioimmunoassay (RIA). In a research hair samples obtained
from morphine treated mice and heroin user contained Nano gram levels of drug
per milligram of hair . The result of the hair analysis for all subject admitting the
use of heroin were positive where as the result of only 30% of thin layer
chromatographic urine analysis of these same subjects were positive.
2) Radioimmunoassay of Hydromorphone & Hydrocodone in Human Plasma
Hydromorphone & Hydrocodone belongs to morphine group of drugs and are
used in combination with antitussive & analgesic antipyretic mixture. The RIA
method is capable of estimating the above drug within a range of 2.5 to 20 ng/mL
using standard 100 µl plasma sample. RIA is carried out using morphine-6-
antiserum & dihydromorphine. Free drug is separated from the bound drug using

235
dextran coated charcoal & an aliquot of the supinate containing the antiserum
bound drug is subsequently counted for radioactivity.
3) Radioimmunoassay of Flunisolide in human plasma Flunisolide is a fast-
acting corticoid designed for the treatment of allergic rhinitis, asthma, and other
allied respiratory disorders in humans. As the quantum of drug delivered by
inhalation (i.e., the usual route of administration of the drug), is invariably small,
the plasma-levels attained can also be fairly small. Hence, there is a need for a
sensitive method of plasma concentration evaluation which is satisfied by
radioimmunoassay.
4) Measurement of Ferritin Serum ferritin levels are indicative of iron stores
present in a patient. Levels are useful in differentiating true iron deficiency from
the body's failure to utilize these stores.
5) Detection of Digoxin This allows direct measurement of serum digoxin
levels quickly and accurately. It is important to rule out Digi toxicity quickly and
accurately. We are also able to filter out Digi bind to let the physician know how
much the level has dropped after Digi bind has been administered.
6) Thyroid Testing This is used to determine the patient's thyroid status and to
follow patients after iodine-131 therapy to see if the dose was indeed effective.
Disadvantages of Radioimmunoassay
• Prolonged reaction time (in days) as a consequence highly diluted reagent
is used.
• Radioisotopes are costly.
• Possible health hazards due to handling of radioisotopes.
• Limited assay range.
• Lack of direct linear relationship between analyte concentration and signal
response.
• Difficult of automation.
• Lengthy counting time.
Surround Optical Fiber Immunoassay
Surround optical fiber immunoassay (SOFIA) is an ultrasensitive, in
vitro diagnostic platform incorporating a surround optical fiber assembly that
captures fluorescence emissions from an entire sample. The technology's
defining characteristics are its extremely high limit of detection, sensitivity,
and dynamic range. SOFIA’s sensitivity is measured at the attogram level
(10−18g), making it about one billion times more sensitive than conventional
diagnostic techniques. Based on its enhanced dynamic range, SOFIA is able to
discriminate levels of analyte in a sample over 10 orders of magnitude,
facilitating accurate titering.

236
 As a diagnostic platform, SOFIA has a broad range of applications. Several
studies have already demonstrated SOFIA’s unprecedented ability to detect
naturally occurring prions in the blood and urine of disease carriers. This is
expected to lead to the first reliable ante mortem screening test
for vCJD, BSE, scrapie, CWD, and other transmissible spongiform
encephalopathies. Given the technology’s extreme sensitivity, additional unique
applications are anticipated, including in vitro tests for other neurodegenerative
diseases, such as Alzheimer's and Parkinson's.]
SOFIA was developed as a result of a joint-collaborative research project
between Los Alamos National Laboratory and State University of New York,
and was supported by the Department of Defense's National Prion Research
Program.
Components of SOFIA
SOFIA comprises a multiwell plate sample container, an automated means
for successively transporting samples from the multiwell plate sample container
to a transparent capillary contained within a sample holder, an excitation source
in optical communication with the sample, wherein radiation from the excitation
source is directed along the length of the capillary, and wherein the radiation
induces a signal which is emitted from the sample, and, at least one linear array.
Steps in SOFIA
Assay preparation
After amplifying and then concentrating the target analyte, the samples are
labeled with a fluorescent dye using an antibody for specificity and then finally
loaded into a microcapillary tube. This tube is placed in a specially constructed
apparatus so it is totally surrounded by optical fibers to capture all light emitted
once the dye is excited using a laser.

237
Figure 1: A schematic representation of SOFIA
Instrumentation processing
This equipment is a spectroscopic (light gathering) apparatus and
corresponding method for rapidly detecting and analyzing analytes in a sample.
The sample is irradiated by an excitation source in optical communication with
the sample. The excitation source may include, but is not limited to, a laser, a
flash lamp, an arc lamp, a light-emitting diode, or the like.
Figure 1 depicts the current version of the SOFIA system. Four linear
arrays extend from a sample holder, which houses an elongated, transparent
sample container which is open at both ends, to an end port. The distal end of the
endport is inserted into an end port assembly. The linear array comprise a
plurality of optical fibers having a first end and a second end, the plurality of
optical fibers optionally surrounded by a protective and/or insulating sheath. The
optical fibers are linearly arranged, meaning that they are
substantially coplanar with respect to one another so as to form an elongated row
of fibers.
Applications
The analyte of interest may be biological or chemical in nature, and by way of
example, only may include chemical moieties (toxins, metabolites, drugs and
drug residues), peptides, proteins, cellular components, viruses, and
combinations thereof. The analyte of interest may be in either a fluid or a
supporting medium, such as a gel.
SOFIA has demonstrated its potential as a device with a wide range of
applications. These include clinical applications, such as detecting diseases,
discovering predispositions to pathologies, establishing a diagnosis and tracking
238
the effectiveness of prescribed treatments, and nonclinical applications, such as
preventing the entry of toxins and other pathogenic agents into products intended
for human consumption:

 Clinical applications – SOFIA may be used to conduct both qualitative tests

(either positive or negative results) to detect or identify bacteria or viruses,
and quantitative tests (measuring substances) to detect or quantify biological
constants or markers, which are substances produced by the body in the
presence of, for example, an infectious disease (to allow determination of viral
load, for instance, in AIDS therapy, or the level of toxicity in drugs of abuse
detection).
 Nonclinical applications - As an immunoassay, SOFIA can potentially be
used on a wider scale to monitor the quality of food, pharmaceuticals,
cosmetics, or water, as well as general environmental parameters and
agricultural products. The ability to detect and screen bacteria and toxins for a
wide range of products is a growing and more complex requirement as may be
evidenced by the increase incidence of food- and animal-borne diseases, such
as E. coli, Salmonella, BSE, avian influenza, etc.
Ante mortem test for prion diseases
SOFIA has been used to rapidly detect the abnormal form of the prion
protein (PrPSc) in samples of bodily fluids, such as blood or urine. PrP Sc is the
marker protein used in diagnostics for transmissible spongiform
encephalopathies (TSEs), examples of which include bovine spongiform
encephalopathy in cattle (i.e. “mad cow” disease), scrapie in sheep,
and Creutzfeldt–Jakob disease in humans. Currently, no rapid means exists for
the ante mortem detection of PrPSc in the dilute quantities in which it usually
appears in bodily fluids. SOFIA has the advantages of requiring little sample
preparation, and allowing for electronic diagnostic equipment to be placed
outside the containment area.
SOFIA as an ante mortem test for prion diseases
SOFIA provides, among other things, methods to diagnose prion diseases
by detection of PrPSc in a biological sample. This biological sample can be brain
tissue, nerve tissue, blood, urine, lymphatic fluid, cerebrospinal fluid, or a
combination thereof. Absence of PrPSc indicates no infection with the infectious
agent up to the detection limits of the methods. Detection of a presence of
PrPSc indicates infection with the infectious agent associated with prion disease.
Infection with the prion agent may be detected in both presymptomatic and
symptomatic stages of disease progression.

239
 These and other improvements have been achieved with SOFIA. SOFIA’s
sensitivity and specificity eliminates the need for PK digestion to distinguish
between the normal and abnormal PrP isoforms. Further detection of
PrPSc in blood plasma has now been addressed by limited PMCA followed by
SOFIA. Because of the sensitivity of SOFIA, PMCA cycles can be reduced, thus
decreasing the chances of spontaneous PrPSc formation and the detection of false-
positive samples.
SOFIA meets the needs of increased sensitivity in the detection of prion
diseases in both presymptomatic and symptomatic TSE infected animals,
including humans, by providing methods of analysis using highly sensitive
instrumentation, which requires less sample preparation than previously
described methods, in combination with recently developed Mabs against PrP.
The method of the present version of SOFIA provides sensitivity levels sufficient
to detect PrPSc in brain tissue. When coupled with limited sPMCA, the methods
of the present inventions provide sensitivity levels sufficient to detect PrP Sc in
blood plasma, tissue and other fluids collected antemortem.
The methods combine the specificity of the Mabs for antigen capture and
concentration with the sensitivity of a surround optical fiber detection
technology. In contrast to previously described methods for detection of PrP Sc in
brain homogenates, these techniques, when used to study brain homogenates, do
not use seeded polymerization, amplification, or enzymatic digestion (for
example, by proteinase K, or “PK”). This is important in that previous reports
have indicated the existence of PrPSc isoforms with varied PK sensitivity, which
decreases reliability of the assay. The sensitivity of this assay makes it suitable as
a platform for rapid prion detection assay in biological fluids. In addition to prion
diseases, the method may provide a means for rapid, high-throughput testing for
a wide spectrum of infections and disorders.
While about 40 cycles of sPMCA combined with immunoprecipitation
were found to be inadequate for PrPSc detection in plasma by ELISA or western
blotting, the PrPSc has also been found to be readily measured by SOFIA
methods. The limited numbers of cycles necessary for the present assay platform
virtually eliminates the possibility of obtaining PMCA-related false-positive
results such as those previously reported (Thorne and Terry, 2008).
Other clinical applications
With rapid developments in the field of biomarker research, many infections
and disorders that have not been possible to diagnose via in vitro testing, are
becoming increasingly possible. SOFIA is predicted to be of broader use in
diagnostic assay development for infections and disorders beyond the scope of
240
prion diseases. A major potential application is for other protein misfolding
diseases, in particular Alzheimer's.
ENZYME LINKED IMMUNOSORBENT ASSAY
The term ELISA was first used by Engvall. ELISA, or enzyme-linked
immunosorbent assay, are quantitative immunological procedures in which the
Ag- Ab reaction is monitored by enzyme measurements. INTRODUCTION TO
ELISA
The ELISA test, or the enzyme immunoassay (EIA), was the first screening
test commonly employed for HIV. It has a high sensitivity. ELISA was
developed in 1970 and became rapidly accepted. An ELISA can be used to detect
either the presence of Antigens or antibodies in a sample depending how the test
is designed. The enzyme converts a colorless substrate (chromogen) to a colored
product, indicating the presence of Ag : Ab binding. Use an enzyme to detect the
binding of antigen (Ag) antibody (Ab).
BASIC PRINCIPLE OF ELISA

Materials Needed:
 Enzyme
 Substrate
 Washing buffer
 Blocking buffer
 Polystyrene microtiter plate
 Antibody (1st, 2nd) / Antigen
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  Testing sample
ANTIGEN (Ag)
Any “thing”, foreign to the immune system. e.g. bacteria, viruses, (or their
parts), pollen, etc. Any molecule that induces production of antibodies when
introduced in the body of an animal is called antigen.
ANTIBODY ( Ab)
Antibody: proteins produced by the immune system which help defend
against antigens

Specimen Sample For ELISA:

 SERUM
 CSF
 SPUTUM
 URINE
 SEMEN
 SUPERNATANT OF CULUTRE STOOL

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243
TYPES OF ELISA
◦ Indirect elisa
◦ Sandwhich elisa
◦ Competetive elisa

244
Sandwich elisa
The antibody of the enzyme conjugate binds with the immobilized antigen to
form a sandwich of antibody- antigen-antibody/enzyme bound to the microwell.
Enzyme reaction product is directly proportional to concentration of standard or
analytical antigen. Antigen in the sample binds with the capture antibody on the
microwell and becomes immobilized. Antigens such as tumor markers, hormones
and serum proteins may be determined

245
Competitive Elisa
Increased serum antigen results in reduced binding of the antigen-enzyme
conjugate with the capture antibody producing less enzyme activity and color
(yellow) formation Substrate product concentration is inversely proportional to
246
the concentration of standard or test antigen added. The bound enzyme conjugate
reacts with the chromogenic substrate added to produce a color reaction (blue to
yellow color). antibody-antigen-enzyme complex bound is inversely related to
the concentration of antigen present in the sample. Serum antigen and labelled
antigen added together--competition. Antibody coated microwell. Used to
determine small molecule antigens.(T3,T4,progesterone etc.)

247
 So incubation, Importance of Washing :- For the removal of any unbound
Antibody/Antigen proper washing and taping is required other wise we get the
incorrect result. During the test performance incubation time and mentioned
temperature is must required For the proper binding between antigen and
antibody and also binding with conjugate and color development of substrate.
Importance of incubation step:- & washing is much important for good results.
Incubate 1 hour .at 37°C 27 Using a clean Pipette , add 100 µL of diluted
serum sample (Dilute the sera to be tested 1:100 in the sample diluents) to each
well 

ELISA PLATE READY FOR READING

Measures the absorbance at 450nm With the help of ELISA READER.
Calculate the absorbance for each sample and reference. We used Ascent
Software for Calculation of the result.
Advantages of ELISA
 Reagents are relatively cheap
 ELISA can be used to a variety of infections.
 Equipment can be inexpensive and widely available.
 Easy to perform and quick procedures
 No radiation hazards occur during labelling or disposal of waste.
 ELISA is highly specific and sensitive
 Have a long shelf life
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Disadvantages of ELISA
 False positives/negatives possible, especially with mutated/altered antigen
 Very specific to a particular antigen. Won’t recognize any other antigen
 Kits are commercially available, but not cheap
 Enzyme activity may be affected by plasma constituents.
 Measurement of enzyme activity can be more complex than measurement
of activity of some type of radioisotopes.
Limitations
 Results may not be absolute
 Antibody must be available
 Concentration may be unclear
 False positive possible
 False negative possible
APPLICATIONS OF ELISA
 Detection of Mycobacterium antibodies in tuberculos
 Detection of rotavirus in feces
 detection of hepatitis B markers in serum
 detection of HIV antibodies in blood samples
 It has also found applications in the food industry in detecting potentialeggs
food allergens, such as milk, peanuts, walnuts, almonds,

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Types
Direct ELISA

Direct ELISA diagram

The steps of direct ELISA follows the mechanism below:

 A buffered solution of the antigen to be tested for is added to each well of

a microtiter plate, where it is given time to adhere to the plastic through
charge interactions.
 A solution of nonreacting protein, such as bovine serum albumin or casein, is
added to well (usually 96-well plates) in order to cover any plastic surface in
the well which remains uncoated by the antigen.
 The primary antibody with an attached (conjugated) enzyme is added, which
binds specifically to the test antigen coating the well.
 A substrate for this enzyme is then added. Often, this substrate changes color
upon reaction with the enzyme.

250
 The higher the concentration of the primary antibody present in the serum, the
stronger the color change. Often, a spectrometer is used to give quantitative
values for color strength.
The enzyme acts as an amplifier; even if only few enzyme-linked antibodies
remain bound, the enzyme molecules will produce many signal molecules.
Within common-sense limitations, the enzyme can go on producing color
indefinitely, but the more antibody is bound, the faster the color will develop. A
major disadvantage of the direct ELISA is the method of antigen immobilization
is not specific; when serum is used as the source of test antigen, all proteins in
the sample may stick to the microtiter plate well, so small concentrations of
analyte in serum must compete with other serum proteins when binding to the
well surface. The sandwich or indirect ELISA provides a solution to this
problem, by using a "capture" antibody specific for the test antigen to pull it out
of the serum's molecular mixture.
ELISA may be run in a qualitative or quantitative format. Qualitative results
provide a simple positive or negative result (yes or no) for a sample. The cutoff
between positive and negative is determined by the analyst and may be statistical.
Two or three times the standard deviation (error inherent in a test) is often used
to distinguish positive from negative samples. In quantitative ELISA, the optical
density (OD) of the sample is compared to a standard curve, which is typically a
serial dilution of a known-concentration solution of the target molecule. For
example, if a test sample returns an OD of 1.0, the point on the standard curve
that gave OD = 1.0 must be of the same analyte concentration as the sample.
The use and meaning of the names "direct ELISA" and "indirect ELISA"
differs in the literature and on web sites depending on the context of the
experiment. When the presence of an antigen is analyzed, the name "direct
ELISA" refers to an ELISA in which only a labelled primary antibody is used,
and the term "indirect ELISA" refers to an ELISA in which the antigen is bound
by the primary antibody which then is detected by a labeled secondary antibody.
In the latter case a sandwich ELISA is clearly distinct from an indirect ELISA.
When the "primary" antibody is of interest, e.g. in the case of immunization
analyses, this antibody is directly detected by the secondary antibody and the
term "indirect ELISA" applies to a setting with two antibodies.

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Sandwich ELISA

A sandwich ELISA.
(1) Plate is coated with a capture antibody;
(2) sample is added, and any antigen present binds to capture antibody;
(3) detecting antibody is added, and binds to antigen;
(4) enzyme-linked secondary antibody is added, and binds to detecting
antibody;
(5) substrate is added, and is converted by enzyme to detectable form.
A "sandwich" ELISA is used to detect sample antigen.[9] The steps are:

1. A surface is prepared to which a known quantity of capture antibody is

bound.
2. Any nonspecific binding sites on the surface are blocked.
3. The antigen-containing sample is applied to the plate, and captured by
antibody.
4. The plate is washed to remove unbound antigen.
5. A specific antibody is added, and binds to antigen (hence the 'sandwich':
the antigen is stuck between two antibodies). This primary antibody could
also be in the serum of a donor to be tested for reactivity towards the
antigen.
6. Enzyme-linked secondary antibodies are applied as detection antibodies
that also bind specifically to the antibody's Fc region (nonspecific).
7. The plate is washed to remove the unbound antibody-enzyme conjugates.
8. A chemical is added to be converted by the enzyme into a color or
fluorescent or electrochemical signal.
9. The absorbance or fluorescence or electrochemical signal (e.g., current) of
the plate wells is measured to determine the presence and quantity of
antigen.
The image to the right includes the use of a secondary antibody conjugated to
an enzyme, though, in the technical sense, this is not necessary if the primary
antibody is conjugated to an enzyme (which would be direct ELISA). However,
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the use of a secondary-antibody conjugate avoids the expensive process of
creating enzyme-linked antibodies for every antigen one might want to detect. By
using an enzyme-linked antibody that binds the Fc region of other antibodies, this
same enzyme-linked antibody can be used in a variety of situations. Without the
first layer of "capture" antibody, any proteins in the sample (including serum
proteins) may competitively adsorb to the plate surface, lowering the quantity of
antigen immobilized. Use of the purified specific antibody to attach the antigen
to the plastic eliminates a need to purify the antigen from complicated mixtures
before the measurement, simplifying the assay, and increasing the specificity and
the sensitivity of the assay. A sandwich ELISA used for research often need
validation because of the risk of false positive results.[10]
Competitive ELISA[edit]
A third use of ELISA is through competitive binding. The steps for this
ELISA are somewhat different from the first two examples:

1. Unlabeled antibody is incubated in the presence of its antigen (sample).

2. These bound antibody/antigen complexes are then added to an antigen-
coated well.
3. The plate is washed, so unbound antibodies are removed. (The more
antigen in the sample, the more Ag-Ab complexes are formed and so there
are less unbound antibodies available to bind to the antigen in the well,
hence "competition".)
4. The secondary antibody, specific to the primary antibody, is added. This
second antibody is coupled to the enzyme.
5. A substrate is added, and remaining enzymes elicit a chromogenic or
fluorescent signal.
6. The reaction is stopped to prevent eventual saturation of the signal.
Some competitive ELISA kits include enzyme-linked antigen rather than
enzyme-linked antibody. The labeled antigen competes for primary antibody
binding sites with the sample antigen (unlabeled). The less antigen in the sample,
the more labeled antigen is retained in the well and the stronger the signal.
Commonly, the antigen is not first positioned in the well.
For the detection of HIV antibodies, the wells of microtiter plate are coated
with the HIV antigen. Two specific antibodies are used, one conjugated with
enzyme and the other present in serum (if serum is positive for the antibody).
Cumulative competition occurs between the two antibodies for the same antigen,
causing a stronger signal to be seen. Sera to be tested are added to these wells
and incubated at 37 °C, and then washed. If antibodies are present, the antigen-
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antibody reaction occurs. No antigen is left for the enzyme-labelled specific HIV
antibodies. These antibodies remain free upon addition and are washed off during
washing. Substrate is added, but there is no enzyme to act on it, so a positive
result shows no color change.
Applications

Human anti-IgG, double antibody sandwich ELISA

Because the ELISA can be performed to evaluate either the presence of
antigen or the presence of antibody in a sample, it is a useful tool for
determining serum antibody concentrations (such as with the HIV test[11] or West
Nile virus). It has also found applications in the foodindustry in detecting
potential food allergens, such as milk, peanuts, walnuts, almonds,
and eggs and as serological blood test for coeliac disease.[13][14] ELISA can
[12]

also be used in toxicology as a rapid presumptive screen for certain classes of

drugs.

Enzyme-linked immunosorbent assay plate

The ELISA was the first screening test widely used for HIV because of its
high sensitivity. In an ELISA, a person's serum is diluted 400 times and applied
to a plate to which HIV antigens are attached. If antibodies to HIV are present in
the serum, they may bind to these HIV antigens. The plate is then washed to
remove all other components of the serum. A specially prepared "secondary
antibody" — an antibody that binds to other antibodies — is then applied to the
plate, followed by another wash. This secondary antibody is chemically linked in
advance to an enzyme.
Thus, the plate will contain enzyme in proportion to the amount of
secondary antibody bound to the plate. A substrate for the enzyme is applied, and
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catalysis by the enzyme leads to a change in color or fluorescence. ELISA results
are reported as a number; the most controversial aspect of this test is determining
the "cut-off" point between a positive and a negative result.
A cut-off point may be determined by comparing it with a known standard.
If an ELISA test is used for drug screening at workplace, a cut-off concentration,
50 ng/ml, for example, is established, and a sample containing the standard
concentration of analyte will be prepared. Unknowns that generate a stronger
signal than the known sample are "positive." Those that generate weaker signal
are "negative".
Dr Dennis E Bidwell and Alister Voller created the ELISA test to detect various
kind of diseases, such as malaria, Chagas disease, and Johne's disease.[15] ELISA
tests also are used as in in vitro diagnostics in medical laboratories. The other
uses of ELISA include:

 detection of Mycobacterium antibodies in tuberculosis

 detection of rotavirus in feces
 detection of hepatitis B markers in serum
 detection of enterotoxin of E. coli in feces
 detection of HIV antibodies in blood samples

Fluoroimmunoassay
Fluoroimmunoassay (FIA) is analogous to RIA except that the label is a
fluorophore rather than a radioisotope. As in other immunoassays, FIA can be
categorized into heterogeneous and homogeneous assays, depending on whether
the separation step is or is not needed, respectively. Either of heterogeneous or
homogeneous assays can be performed in a competitive or non-competitive
format. Heterogeneous competitive FIA methods are currently available for
analysis of various compounds of pharmaceutical importance in biological fluids:
aminoglycoside antibiotics, morphine-3-glucuronide, the major urinary
metabolite of heroin and morphine, benzoylecogonine in urine, and endocrine
disruptTing chemicals (estrone, estradiol, and ethinylestradiol) in a complex
synthetic aqueous matrix . These FIA offered sensitivities ranged from 0.01-2
ng/ml. A heterogeneous competitive FIA approach was developed for the
measurement of the fluorescent signal directly on the solid supports, wells of
microwell plates. This approach was used for the determination of the total
thyroxine in human serum. In a further work, the fluoTrescence signal measured
directly onto the solid phase was considerably stabilized by pretreatment of the
wells with a glycerin solution. This treatment caused in an increase in the
sensitivity of the assay. In homogeneous FIA, the antibody-bound analyte is not
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needed to be separated from the free analyte before the fluorescence
measurement. Almost all the homogeneous FIA applied in pharmaceutical
analysis are performed as competitive. In these assays, the antibody binding
causes some changes in the fluorescence properties (e.g. polarization) of the
labelled analyte. The analyte concentration in a sample can be monitored directly
from the reaction mixture. The most common type of these assays is the
fluorescence polarization fluoroimmunoassay (FPFIA). This technique is based
on the following: when a fluorescent analyte conjugate is excited with polarized
light, the polarization of the resulting emission depends inversely on the decay
constant of the probe (4-5 ns for fluorescein isothiocyanate) and on the rotational
motion of the conjugate. With small molecules (e.g. drugs), random rotation
decreases the polarization signal; when bound to specific antibodies, their
rotation slows, and the polarization signal increases. The increase in the
polarization signal is related to the concentration of the analyte. Originally, this
technique was developed for single tube analytical instruments, but the
technology was rapidly converted Immunoassays into high-throughput screening
assays when automated analyzers became commercially available. The FPFIA
methods utilizing automated analyzer are simple, precise, and easy to perform.
Therefore, these methods are widely applied in drug discovery studies as well as
in therapeutic monitoring of a wide range of analytes, shown in Table Table5.5.
Recently, an improved one-step FPFIA was developed for analysis of
progesterone hormone using an immunocomplex single reagent (a pre-
equilibrated mixture of antibody and tracer). This assay is a speedy and does not
need incubation period before the measurement of the fluorescence polarization
signal (the total assay time is about 7 min for 10 samples). The limit of detection

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of this assay was 2.7 ng/ml with 50 ml samples.

Time-resolved fluoroimmunoassay (TRFIA) is a separate group of FIA because

its principles can be adapted to both heterogenous and homogenous assay format.
In TRFIA, chelates of lanthanides and palladium ions are used as labels . Highly
sensitive TRFIA methods were developed for analysis of many pharmaceutical
compounds and hormones in biological fluids (Table 6). The advantage in these
assays was the possibility to resolve the background fluorescence (due to
biological fluids) from the assay fluorescence. Therefore, the non-specific
endogenous fluorescence was eliminated.

Chemiluminescence Immunoassay
Chemiluminescence immunoassay (CLIA) involves a chemiluminescent
substance as a label. The growing success of this technique in pharmaceutical
analysis due to its high performance, low detection limits, and good precision.
The principles, methodology, instrumentation, and applications of
chemiluminescence immunoassay are the subject of some books and reviews.
The CLIA was used for analysis of many compounds of pharmaceutical
importance; these compounds are shown in Table 7.

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Table 7
List for Compounds of Pharmaceutical Importance Analyzed by
Chemiluminescence Immunoassay

Normal results
Immunoassays which are qualitative are reported as positive or negative.
Quantitative immunoassays are reported in mass units, along with reference
intervals (normal ranges) for the test. Normal ranges may be age- and gender-
dependent. Positive immunoassay test results for HIV and drugs of abuse
generally require confirmatory testing.
Although immunoassays are both highly sensitive and specific, false
positive and negative results may occur. False-negative results may be caused by
improper sample storage or treatment, reagent deterioration, or improper washing
technique. False-positive results are sometimes seen in persons who have certain
antibodies, especially to mouse immunoglobulins (immune cells) that may be
used in the test. False-positive results have been reported for samples containing
small fibrin strands that adhere to the solid phase matrix. False-positives may
also be caused by substances in the blood or urine that cross-react or bind to the
antibody used in the test.
Applications:
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 Immunoassay techniques using the highly specific and sensitive nature of
immunological reactions have been developed and applied in the food industry
for detecting the naturally occurring constituents, antibiotics, pesticide residues,
microorganisms, and fragments of microbial constituents related to food analysis,
food production, food processing, and food safety. Both polyclonal and
monoclonal antibodies are employed for the development of the various
immunoassay systems, including enzyme-linked immunoassay (ELISA) and
radioimmunoassay (RIA). Immunoassay techniques provide complementary
and/or alternate approaches in reducing the use of costly, sophisticated
equipment and analysis time, but still maintaining reliability and improved
sensitivity. Immunoassay techniques in their most simple forms provide excellent
screening tools to detect adulteration and contaminations qualitatively. The
application of immunoassay techniques contributes tremendously to the quality
control and safety of our food supply.
Immunoassays can be used to test for the presence of a specific antibody or
a specific antigen in blood or other fluids.
When immunoassays are used to test for the presence of an antibody in a
blood or fluid sample, the test contains the specific antigen as part of the
detection system. If the antibody being tested for is present in the sample, it will
react with or bind to the antigen in the test system and will be detected as
positive. If there is no significant reaction, the sample tests negative. Examples of
immunoassay tests for antibodies include Rheumatoid Factor (which tests for the
presence of autoimmune antibodies seen in patients with rheumatoid
arthritis), West Nile Virus (which tests for antibodies that a person made in
response to an infection with that virus) or antibodies made in response to
a vaccination (such as tests for antibodies to Hepatitis B to assure that the
vaccination was successful).
When immunoassays are used to test for the presence of antigens in a blood
or fluid sample, the test contains antibodies to the antigen of interest. The
reaction of the antigen that is present in the person's sample to the specific
antibody is compared with reactions of known concentrations and the amount of
antigen is reported. Examples of immunoassay tests for antigens include drug
levels (like digoxin, vancomycin), hormone levels (like insulin, TSH, estrogen),
and cancer markers (like PSA, CA-125, and AFP).

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