COMPANY PROFILE

SANJIVANI PARENTERAL LIMITED is a research-based, international pharmaceutical

company that provides a wide range of high quality products & services, at affordable
prices. The core product range of the company’s products includes oral solids, small
volume PARENTERALS and sterile powder formulations. Established in 1997 the
Company has grown many folds and aspires to be a respected research-based company.
With its diverse product portfolio, it is rapidly expanding its reach to the global
marketplace, riding on its success in India and in the world’s emerging and developed
markets. SANJIVANI is the largest supplier of medicine in Indian ARMY hospitals,
Indian railway Hospitals, ESIC hospitals, and so many institutions.
It has 3 WHO-GMP certified manufacturing units in India.
Mumbai- The company has state-of-art WHO-GMP approved manufacturing facility at
Navi Mumbai (New Mumbai). It was incorporated in 1997. It produces oral solids,
capsules, syrups and injections.
The manufacturing capacity of this plant is about 6.5 million vials/ampoules per month.
People working at the plant change their clothes and enter the plant in the clothes that are
specially designed and cleaned to suit the requirements of the ambience of the plant, to
avoid any sort of contamination.
Dehra Dun-
The company has started an oral solid plant in Dehra Dun and begun manufacturing
tablets and capsules. It has capacity of manufacturing 80 millions tablets per month and
25 million capsules per month.
During the year 1998-99, the company has been approved by the WHO-GMP Certificate.
The company is hoping for export its products in Africa, Europe & Latin America.
The company has tied up with Cadila Ltd., Alkem Ltd., Indian Immunologicals Ltd for
marketing its products.
During the year 1999-2000, the company has tied-up with India’s first ranked
pharmaceutical giant Ranbaxy for manufacturing its products.

1
 INTRODUCTION

Now a days, there are several companies manufacturing drugs; but a very few among
them are providing effective drugs to the society at affordable price. Elder
Pharmaceutical is one of the manufacturers of different effective drugs which are saving
life throughout not only India but abroad also. Its drugs are not beyond the capacity of
lower section of society, while retaining good quality.

Elder Pharmaceutical started its Pharma business in 1959. The entire group was
conceived as small generic pharmaceuticals company in 1971 by Mr.U.N.Mehta, Late
founder chairman. Starting with anti-rheumatic and CNS drugs, Torrent discovered an
untapped opportunity in marketing medicines to specific segments. Elder‘s first
manufacturing facility was set up in Ahmedabad (Gujarat) in early 80s. Its first export
order was held in 1983. Elder Pharmaceutical grew its pharma business on back of
exports to USSR. New manufacturing facility commissioned at BADDI (Himachal
Pradesh) in November 2005. Its market has reached across 50 countries, along with 3000
strong sales and marketing team globally.

Elder Pharmaceutical is amongst the largest spenders on R & D (9% of sales per
annum). It has collaborate research with Astra Zeneca. It is only Indian contract
manufacturer of Insulin for Novo Nordisk since 1990. Current it is ranked 14 th in India by
ORG-MARG, based on domestic sales.

Elder Pharmaceutical has started a new state of the art formulations manufacturing
facility at Baddi, Himachal Pradesh 45 kms from Chandigarh city. The total plot area is
25 acres (82238 square meters). It has a capacity to manufacture 3600 million tablets and
capsules per annum. It commenced its operation in November 2005.

TYPES OF PRODUCT MANUFACTURED:

 SOLID ORAL DOSAGE FORMS

Tablets

Capsules

 LIQUID ORAL DOSAGE FORMS

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 Suspension /syrups

The total products have an international acceptability of results that provides

recognition to the laboratory and in turn ensures the performance of the laboratory, in
accordance with international standards. This has minimized the risk of unreliable results
and reduces the chances of retesting. Elder Pharmaceutical established the concept of
“tested once and accepted everywhere”.

Elder Pharmaceutical Industry Ltd is registered as pharmaceutical company in Gujarat.

Elder Pharmaseutical has established its Quality System in accordance with
ISO/IEC17025:2005. The Quality system is developed based on the 4 Ms (Man,
Machine, Material and Method of testing) or A (accommodation conditions i-e
environmental suitability).

Elder Pharmaceutical has different divisions or departments. They are:

Quality department – it has two branches

 Quality control

 Quality assurance

Quality Control (QC) Department :-

It is a process through which a microbiologist ensures that product quality is maintained

or improved and manufacturing errors are reduced or removed.

Or in simple words we can say that-

“It is part of quality management focused on fulfilling quality requirements.”

It works in two parts:-

 Chemical and instrumentation: here sampling and analysis of raw

materials, analysis of intermediates and finished products are done.

 Microbiology: here sampling and testing of raw material for microbiological

tests and water testing, environmental monitoring, validation of various microbiological
aspects.

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 Quality Assurance (QA) Department:-

Quality Assurance is a way of preventing mistakes and defects in manufactured products and
avoids problems when delivering the final products.
It is a procedure that focuses on providing assurance that quality request will be achieved.

4
 INTRODUCTION

Since the time of Antony Van Leeuwenhoek who first observed the microbes, the micro
biology is developing day by day and creating milestones in areas relating to human
health, wealth and prosperity. Louis Pasteur and Robert Koch gave the new trends to
microbiology and had contributed a lot to human development.

Nowadays microbiology is not only the study of diseases but it also finds
application in industry in the production of enzymes, antibiotics, vitamins, drugs ,
processed foods and fermentation industries.

On one hand microbes are need of many industrial processes but on the other
hand these are restricted in some manufacturing processes because they may cause
serious contamination which can affect the equality of product.

In drug manufacturing plants the presence of microbes is specially restricted as

the presence of any microbe or pathogen may have very severe outcomes. Contamination
at any level in production of injectible and orally administered drugs may pose threat to
human life.

Therefore to maintain the quality of drugs we perform different tests like microbial
limit test etc.

In this project we will discuss the microbiological aspects required to maintain and
assure the quality of drugs.

The fundamentals of quality, quality control, quality assurance

 Quality:-according to Philip B. Crossby “Quality is meeting the requirements”

In the pharmaceutical industry the requirements involve meeting specifications and

developing system to make certain that materials are sampled, tested and evaluated
against standard specifications.

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QUALITY CONTROL:-

The aggregate of activities such as design of analysis, statistical sampling, and inspection
for defect are designed to ensure adequate quality of manufacturing product. For
pharmaceutical industry quality control department performs these functions. The quality
department evaluates the materials against the requirements and defines the quality of
product.

Quality control has become a separate discipline in the pharmaceutical industry. Still
Quality control doesn’t ensure the quality of product in the strictest manner as done by
the manufacturing department. The quality department only monitors the product and
production operations. The laboratory controls all the records and maintains security,
confidentiality of these records and ensures that these documents can be retrieved as and
when required. The laboratory ensures the protection of back up records and prevention
of unauthorized access. The factor determining the correctness and reliability of results is
taken into account by the laboratory. Personnel performing specific task are qualified on
the basis of appropriate education, training experience and/or demonstrated skills. All the
methods are validated, indicating that the particular requirements for the intended use are
fulfilled.

Before being placed into service the instrument or equipment is calibrated to establish
that it complies with the relevant standard specifications. The laboratory performs
intermediate checks to provide extra assurance to instruments performance.

The laboratory has the procedure for handling, protection, storage and disposal of test
samples, calibration of equipments to protect the interest of laboratory. The performance
of each test and calibration is carried out by laboratory personnel. The results are reported
accurately, clearly, unambiguously, objectively and in accordance with any specific
instruction in the test and calibration methods.

QUALITY ASSURANCE:-

The quality of product depends on more than just sampling, testing and release of its
various components. it is a means of ensuring that the facilities and equipments are in
order and that the production department was adequately functioning and the systems in

6
the process are adequate. The processes are documented and the quality control
department itself does its job adequately and completely, this overall concept of quality
has been come to be known as total quality control and more recently as quality
assurance.

Quality assurance is an overall company wide concept that ensures the establishment of
certain factors during production, the evaluation of certain results after production. The
quality assurance department has different divisions

 Microbial

 Chemical and instrumentation

 In process quality assurance

 Stability

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 MICROBIAL LIMIT TEST (MLT)
These tests are used for estimation of the number of viable aerobic micro-organisms
present and for detecting the presence of microbial species in the pharmaceutical
substances.

TOTAL MICROBIAL COUNT:-

Total microbial count has two divisions:

(A) BACTERIAL COUNT

(B) FUNGAL COUNT

(A) BACTERIAL COUNT

Medium used: SOYBEAN CASEIN DIGEST AGAR (SCDA)

SAMPLE PEPERATION:

About 10gm or 10ml of the preparation being examined is dissolved in sterile buffered
sodium chloride peptone solution pH-7 and the volume is made up to 100ml.

In case of insoluble products certain surface active agents are added such as 1gm/l of
Polysorbate 80 to assist the suspension of poorly wettable substances.

PROCEDURE:

1ml of the sample solution was transferred into 9ml of liquefied (45ºC) Soybean Casein
Digest Medium in the test tube. It gave 1:10 dilution of the original sample solution.

In the next step, 1ml of 1:10 dilution was taken and added to 9ml of same medium. This
gave 1:100 dilution of the original sample solution.

1ml of each sample dilution (10-1 and 10-2) was transferred into two sterilized Petri
plates.

About 20ml of liquefied Soybean Casein Digest Agar was added which is not more than
45ºC.

The plate was swirled and allowed to solidify.

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 Plates were incubated at 30ºC - 35ºC for 5 days unless a reliable count was obtained in
a shorter time.

The number of colonies formed was counted and the average was expressed for the two
plates in terms of number of micro-organisms per gm or ml of the substances.

CALCULATION:-

Bacterial count, cfu per gm or ml= average number of colonies X dilution factor

If no colonies were recovered from the dish representing the initial 1:10 dilution of the
substances, the result was expressed as “less than 10 micro-organism per substance”.

(B) FUNGAL COUNT

Medium used: SABOURAUD’S DEXTROSE AGAR

PROCEDURE:

 About 1ml of each sample dilution (10-1 and 10-2) was transferred into two sterile
Petri plates.

 0.005% of chloramphenicol was added to SDA medium at not more than 45ºC
and then about 20ml of media was added to the Petri plates.

 The plates are swirled to mix and allowed to solidify.

 The plates were incubated at 22.5ºC- 25ºC for 5 days.

 The colonies formed were counted.

CALCULATION:

Fungal count cfu/gm or ml = average number of colonies X dilution factor

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PATHOGEN IDENTIFICATION TEST
(A) Escherichia coli

Prescribed quantity of sample was transferred to a sterile flask containing 50ml

of nutrient broth. The flask was shaken and allowed to stand for 1 hour (4 hours for
gelatin) and shake again. Incubation was done at 36ºC-38ºC for 18-24 hours.

PRIMARY TEST:-

About 1ml of enrichment culture preparation was added to a tube containing

5ml of MacConkey’s broth followed by the incubation in the water bath at 36ºC-38ºC for
48 hours. Simultaneously MacConkey agar media was also streaked with the enrichment
culture preparation to observe Brick red colonies of gram negative rods. Secondary test
could be carried out if acid, gas production and brick red colonies appear in the content of
the tube, otherwise the sample meets the requirements of the test for the absence of
E.coli.

SECONDARY TEST:-

0.1ml of content of above tube was added to the tube containing:

(a) 5ml of MacConky’s broth and

(b) 5ml of peptone water.

Incubation was done in the water bath at 43.5ºC-44.5ºC for 24hours and tubes were
examined for:

(a) Acid and gas

(b) Indole

To test for indole, 0.5ml of kovac’s reagent was added, shaked and allowed to stand for
1 minute. Appearance of red color in the reagent layer confirms the presence of indole.
The presence of acid, gas and indole in the secondary test confirms the presence of E.coli.

Secondary testing also involves the streaking of enrichment medium on Levine Eosin
Methylene Blue gar media and observed for colonies with metallic sheen under reflected
light and a blue black appearance. Simultaneously a positive test was also carried out by

10
repeating the primary test by adding 1.0ml of the enrichment culture and a volume of
broth containing E.coli, prepared from a 24 hours culture in nutrient broth to 5ml
MacConky’s broth. The test not valid unless the results indicate that the control contains
E.coli.

(B)Salmonella

ENRICHMENT CULTURE PREPERATION:-

Prescribed quantity of sample was transferred to a sterile flask containing 100ml of

nutrient broth, shaked and allowed to stand for 4 hours and again shaked. Incubation was
given for 24 hours at 35 ºC- 37ºC temperature.

PRIMARY TEST:-

1ml of the enrichment culture preparation was added to each of the two tubes containing
10ml of:

(a) Selenite F broth

(b) Tetrathionate -bile –brilliant green broth

And incubated at 36ºC-38ºC for 48 hours. From each of these two cultures, subculture
was done on at least two of the following four agar media;

(a) Bismuth Sulphite Agar

(b) Brilliant Green Agar

(c) Deoxycholate Citrate Agar

(d) Xylulose Lysine Deoxycholate Agar.

The plates were then incubated at 36ºC for 18-24 hours. Upon examination, if none of the
colonies confirms to the description given in table below, the sample meets the
requirements of the test for the absence of the genus Salmonella.

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TABLE- Description of Salmonella

MEDIUM DESCRPTION OF COLONIES

1) Bismuth Sulphite Agar Black and green

2) Brilliant Green Agar Small, transparent and colorless or opaque

Colorless and opaque with or without

3) Deoxycholate Citrate Agar
centers

4) Xylulose Lysine Deoxycholate Agar Red, with or without black centers.

Any colonies confirming to the description given in table obtained, secondary test was
carried out:

SECONDARY TEST:-

Any colony showing characteristic given in the table was sub cultured in Triple Sugar
Iron Agar slant by first inoculating the surface of the slope and then making a stab culture
with the same inoculating needle, and at the same time a tube of Urea broth was also
inoculated. Incubation was done at 36 ºC to 38ºC for 18-24 hours. The formation of acid
and gas in the stab cultures (with or without concomitant blackening) and the absence of
acidity from the surface growth in the TSA slant together with the absence of red color in
the urea broth, indicate the presence of Salmonella.

Simultaneously a control positive test was also carried out by repeating the primary and
secondary test using 0.1ml of enrichment culture and a volume of broth containing
Salmonella (standard organism) prepared from 24 hour old culture in nutrient broth. The
test is not valid unless the results indicate that the control contains Salmonella.

(C) Pseudomonas aeruginosa

ENRICHMENT CULTURE PREPERATION:

Prescribed quantity of sample was transferred to a sterile flask containing 100mlof

Fluid Soybean Casein Digest Medium, shaked and incubated at 35-37ºC for 24 to 48
hours. A portion of Cetrimide Agar plates with the above preparation. If upon
examination, none of the plates contain colonies having the characteristics listed in table

12
below for the media used; sample meets the requirements of the test for absence of
Pseudomonas aeruginosa.

TABLE: Description Of Pseudomonas aeruginosa

Media Description of Fluorescence in Oxidase test Gram stain

colonies U.V light

Cetrimide agar Generally Greenish Positive Gram negative

medium greenish rods

Pseudomonas
agar medium for Colorless to Yellowish Positive Gram negative
detection of yellowish rods
fluorescence

Pseudomonas
agar medium for Generally Blue Positive Gram negative
detection of greenish rods
pyocyanin

Oxidase test was only carried out when ay colony confirming to the description given in
the table were produced:

Pigment test:

Suspected colonies were streaked from the agar surface of Cetrimide agar on the
surface of Pseudomonas agar medium for detection of fluorescence and Pseudomonas
agar medium for detection of pyocyanin. Incubation was done at 33ºC-35ºC for three
days.

The plates were examined to determine whether colonies confirming to the description
in the table above were present.

A positive control was also carried out.

Oxidase test:-

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 This test was carried out after the appearance of the suspected colonies. It was done by
placing 2 or 3 drops of a freshly prepared 1% w/v solution of N,N,N,N,-tetramethyl-4-
phenlenediamine dihydrochloride on filter paper and smear with the colonies; the sample
met the requirement only when there was no development of a pink color changing to
purple, showing the absence of Pseudomonas aeruginosa.The appearance of purple color
within 15 minutes gives the positive results.

(D) Staphylococcus aureus:

ENRICHMENT CULTURE PREPERATION:-

Prescribed quantity of the sample was transferred to a sterile flask containing 100ml of
Fluid Soybean Casein Digest Medium, shaked and incubated at 35ºC-37ºC for 24 to 48
hours. This was then streaked on the surface of one of the media listed in the table. The
plates were then incubated at 35ºC-37ºC for 24-48 hours. To meet the requirements of the
absence of Salmonella, the must not contain any colony.

TABLE: Detection of Staphylococcus aureus

Selective media Characteristic morphology of Gram stain

colonies

Positive cocci (in

Vogel-Johnson media Black surround by yellow zones
cluster)

Positive cocci (in

Mannitol salt agar Yellow colonies with yellow zones
cluster)

Black, shiny surrounded by clear zones Positive cocci (in

Baird parker agar
of 2 -5 mm cluster)

Coagulase test was carried out on the occurrence of the growth.

Representative suspected colonies from agar surface of any of the media were transferred
to the individual tubes each containing 0.5ml of mammalian plasma with or without
additives. Incubation was done in water bath at 37ºC examining the tubes at 3 hours and
subsequently at suitable intervals upto 24 hours. The non- occurrence of coagulation
meets the requirements of the absence of Staphylococcus aureus.

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Simultaneously positive and negative control was also carried out.

RESULTS:-

SAMPLE -- VALZAR 40

Total viable aerobic count:-

Sample preparation:-

10g of sample was taken and transferred in to 90ml Fluid Casein Digest Soy Lecithin
polysorbate20 Medium.

Total bacterial count :-

1ml of the above solution transferred in Petri dish of Soybean Casein digest Agar
Medium in Duplicate with incubation at 32.5ºC for 24 hours.

Calculation:-

Bacterial count per gm= mean value of colonies observed X dilution factor

= 6 X 10

= 60

Test for fungal count:-

1ml of the sample preparation transfer into Petri dish of Sabourauds Dextrose Agar
Medium in duplicate with incubation at 22.5ºC for 48 hours.

Calculation:-

Fungal count per count= mean value of colonies observed X dilution factor

= nil

Result:-

Total viable aerobic count (cfu/gm) = bacterial count + fungal count

= 60

Pathogen test:-

Test for E.coli and Salmonella

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Pre- enrichment-

Weight of sample= 10gm

Media used = Fluid lactose medium 90ml

Incubation temperature = 32.5 ºC

Test for E.coli:-

Primary testing:-

Media used= MacConky agar media and MacConkys broth

Observation= no acid and gas production and Brick red colonies of gram

Negative rods absent.

Secondary testing:-

Media used= MacConkys broth, peptone water, kovac’s reagent and Levine

Eosin methylene blue agar media.

Observation= no red color and growth was observed.

Test for Salmonella:-

Primary testing:-

(a)Volume of pre-enriched media= 1ml

Media used= 10ml of Fluid Cysteine Medium

10ml of Fluid tetrathionate Medium

Incubation temperature= 32.5ºC

Incubation time= 24 hours

(b) Media used= Brilliant Green Agar Medium

Bismuth Sulphite Agar

Incubation temperature= 32.5ºC

Incubation time= 24 hours

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 Observation:- no growth and colonies were observed.

Secondary testing:-

Media used= Triple iron sugar agar

Observation= no alkaline and acid butts were observed

Test for Pseudomonas aeruginosa and Staphylococcus aureus:-

Pre-enrichment:-

Weight of sample= 10gm

Media used = Fluid Soybean Casein Digest Medium.

Incubation temperature= 32.5ºC Incubation time= 24 hours

Test for Pseudomonas aeruginosa:-

Primary testing:-

Media used= Cetrimide Agar Media

Incubation temperature= 32.5ºC

Incubation time= 48 hours

Observation= no greenish gram negative rods were observed.

Test for Staphylococcus aureus:-

Primary testing:-

Media used= Mannitol Salt Agar Medium

Incubation temperature= 32.5ºC

Incubation time= 48 hours

Observation= no yellow colonies with yellow zones were observed.

Every test has to be accompanied by a positive and negative control.

The positive control must be consisting of the medium containing the sample and the
pure culture of the concerned organism. This should necessarily show a positive result.

17
 The negative control must be consisting of only the medium. After incubation, it should
necessarily show a negative result.

The positive and negative controls are kept to ensure the validity of the test.

Table:-MLT Results of Various Finished Products

TBC TFC E.coli Salmonella Pseudomonas Staphylococcus

Sample
abony aeruginosa aureus

Alprax forte 35 Nil absent Absent absent absent

Domstal
20 Nil absent Absent absent absent
suspension

Azona 40 30 Nil absent Absent absent absent

Ranitin 300 20 Nil absent Absent absent Absent

Omizac 20 Nil absent Absent absent Absent

Dilogesic 30 Nil absent Absent absent Absent

All the values are well with in the specified limits (limit NMT 100 cfu /gm or ml). Hence,
the products are of standard quality.

0Table:-A comparative study was also done with different batches of Enzystal tablets.

TBC TFC E.coli Salmonella Pseudomonas Staphylococcus

Sample
abony aeruginosa aureus

Batch no.1 80 Nil absent Absent absent Absent

Batch no.2 60 Nil absent Absent absent Absent

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Batch no.3 70 Nil absent Absent absent Absent

Batch no.4 60 Nil absent Absent absent Absent

Batch no.5 80 Nil absent Absent absent Absent

Batch no.6 70 Nil absent Absent absent Absent

Batch no.7 70 Nil absent Absent absent Absent

Batch no.8 50 Nil absent Absent absent Absent

Batch no.9 60 Nil absent Absent absent Absent

Batchno.10 70 Nil absent Absent absent Absent

Graph:-This comparison was well studied with the help of graph:

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MORPHOLOGICAL TESTING:-

GRAM STAINING:-

Gram staining is a procedure of classifying micro organism as gram negative

and gram positive .The gram positive bacteria appear purple after staining
procedure and gram negative bacteria appear red.

PROCEDURE:-

 Wear sterile gloves and mask.

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  Take clean dry glass slide and make it grease free by passing it through flame 3- 4
times.

 Take the colony to be identified by means of sterile inoculating loop.

 Prepare smear and allow it to air dry.

 Gently pass it through flame 2-3 times to heat fix the smear.

 Allow the slide to cool and then flood it with water.

 Stain the slide with crystal violet for one minute and then wash it with water.

 Flood the slide with gram’s iodine for one minute, these acts as a mordent. Wash
the slide with water.

 Decolorize the stained slide by flooding it with alcohol .Allow it to stand for 30
seconds. Wash again with water.

 Again stain the slide with 0.5% safranin for 1 minute.

 Wash the slide with water and allow it to air dry.

 Observe the slide under microscope and note down cell shape and color.

 Violet colored cells represent gram positive cells and red cells represent gram
negative cells.

SHAPES:-

 Coccus (single spherical cells)

 Diplococci-cocci in pairs

 Streptococci –cocci forming chains

 Tetrads –cocci forming squares

 Sarcinae- cocci forming cubes

 Staphylococci- cocci forming amorphous sheets or clumps.

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 Baccilus (red shaped cell)

 Bacilli -single rod

 Diplobaccilli- bacilli in pairs

 Streptobaccilli- bacilli in chains of cells

 Coccobacilli- cocci like bacilli

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WATER ANALYSIS
Water is the most widely used substances, raw material, or ingredient in the production,
processing and formulations of the compendial articles. Control of the microbiological quality
of these waters is important because proliferation of these micro-organisms ubiquitous to water
may occur during the purification, storage and distribution of this substance. If water is used in
the final product, these micro-organisms or their metabolic products may eventually cause
adverse consequences.

Water that is used in the early stages of the production of drug, substances and that is the source
or feed water for the preparation of the various types of purified waters must meet the
requirements of the National Primary Drinking Water Regulations (NPDWR) (40 CFR141)
issued by the Environmental Protection Agency (EPA). These requirements ensure the absence
of coliforms, which, if determined to be of faecal origin, may portend or indicate the presence
of other micro-organism of the faecal origin, including viruses that may be pathogenic for
humans. For this purpose, there are many different grades of pharmaceutical waters.

TYPES OF WATER:-

1. Drinking water

2. Processed water

3. Purified water

4. Water for injection

1. DRINKING WATER:-

Drinking water is not covered by compendial monographs but must comply with the quality
attributes of the EPA, NPDWR or comparable regulations of the European Union or Japan. It
may be derived from a variety of sources, including a public water utility, a private water
supply (e.g. a well) or combination of more than one of these sources. Drinking water may be
used in the early stages of chemical synthesis and in the early stages of the cleaning of

23
pharmaceutical manufacturing equipment. It is the prescribed source feed water for the
production of pharmaceutical waters.

2. POTABLE WATER OR PROCESSED WATER:-

Potable water is generated through following sequence:

Water is pumped to Raw Water Storage Tank through bore wells. Continuous overflow has
been provided to avoid stagnation of water in Water Storage Tank. Two Raw Water Tanks have
been provided to avoid interrupted water supply in case if cleaning of tank is in process.

This water is then treated with Sodium Hypochlorite to maintain chlorine content of 2ppm in
the water. This is done to inhibit the bacterial growth in water storage tank.

This water is then passed through Dual Media Filter or Multigrade filter to remove suspended
particle and non-colloidal silica. Dual media filter is filled with gravels and sand.

Again water is treated with Sodium Metabisulphite to reduce chlorine content to acceptable
levels. SMBS dosing is controlled and residual chlorine is measured by online chlorine
Analyzer and then stored in Filter Water Storage Tank.

24
This filtered water is pumped in to water softeners. Water softeners remove cations such as
calcium and magnesium that interfere with the performance of downstream processing
equipments. Water softeners resin beds are regenerated with sodium chloride solution (brine).
Softeners are with Food Grade resins to avoid leachable in the potable water system. These
softeners are designed to provide water with commercial hardness zero.

Table:-This potable water has following specification:

PARAMETERS VALUES

Ph 6.5 to 8.5
Total Dissolved Solids 500mg/l max.
Calcium 75 ppm max
Magnesium 30 ppm max
Iron 0.3 ppm max
Manganese 0.1 ppm max
Chlorine 250 ppm max
Sulphate 200 ppm max
Nitrite 45 ppm max
Fluoride 1.0 ppm max

This potable processed water is pumped to two potable storage tanks in the production
blocks. One tank provides water for further purification to get purified water and another tank
provides water for cooling towers. Processed water distribution is through closed loop with
water circulating at the rate of 1 meter per second to avoid stagnant water and have turbulent
flow to minimize Bio Film formation. This will help to minimize bacterial growth in the
process water loop, minimize the burden on the purified water system and also on final water
rinses. However no loop has been provided to the point of use. This avoids the stagnation of
water in the branch connection.

3. PURIFIED WATER:-

25
 Pretreated water goes into Multi-grade Filter. The filtering media is granular made of
anthracite, quartz and sand. Granular or cartilage filter are used for pre-filteration. They remove
the solid contaminants from the water supply and protect downstream system.

This water is then passed through softners to remove the hardness of water. Softners utilize
sodium based cation exchange resins to remove water hardness ions, such as calcium and
magnesium that could foul or interfere with the performance of downstream processing
equipment such as reverse osmosis membranes, deionization devices and distillation units.
Water softners can also be used to remove other lower affinity cations, such as the ammonium
ions, that may be released from chloraminedisinfectants commonly used in drinking water.
Water softner beds are regenerated with concentrated sodium chloride solution (brine)

Now the water is passed through a 5µm cartilage filter.

This water is then passed through Heat Exchanger to maintain the temperature. This heat
exchange system consists of two pipes placed adjacent to each other. In one pipe soft water
flows and in another chilled water. Chilled water lowers down the temperature of water to
25ºC.

This water is treated with Sodium Metabisulphite and then with 10% NaOH.

Water is then passed through Rota meter to maintain the flow rate.

26
 Again water is passed through softners and from 5 micron cartilage filters

This water is then stored in Feed Tank, from which by high pressure pump waters passed
through RO system (reverse osmosis). RO units employ semi permeable membranes. The
‘pores’ of RO membranes are actually intersegmental spaces among the polymer molecules.
They big enough for permeation of water molecules, but too small to permit the passage of
hydrated chemical ions. RO units can be used alone or combination with DI units as well as
ultrafilteration for operational and quality enhancement.

After RO system water moves to Digaser (to especially remove carbon dioxide)

Now water moves to CDI system (continuous electron deionization). It is an effective method
of improving the chemical quality attributes of water by removing the cations and anions. CDI
system has charged resins that require periodic regeneration with an acid and base. Typically
cationic resins are regenerated with either hydrochloric acid or sulphuric acids, which replace
the captured positive ions with hydrogen ions. Anionic resins are regenerated with sodium or
potassium hydroxide which replaces captured negative ions with hydroxide ions. The system
is designed in such a way that both the cations and anions are mixed together to form a mixed
bed.

Then water is then stored in Purified Water Storage Tank (3000l) and then water goes to the
distribution system.

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METHODS OF ANALYSIS:-

There are three methods of analysis:

Membrane Filteration Method

Pour Plate Method

Most Probable Number Method

Membrane Filteration Method

In this method the cellulose nitrate filter having a pore size not more than 0.2µ diameter about
47mm is used. Membrane filter unit is assembled by inserting the filter holder into the neck of
the flask and sterile filter disk is placed on the holder base. Side arm of the flask is attached to
the vacuum pump through a rubber pipe and measured quantity of water is poured in the
funnel. Generally 50ml of water is used for filtration. The vacuum pump is put on and water is
filtered. After filtration the vacuum pump is disconnected, the funnel is removed and filter disc
is carefully taken out with the help of sterilized forceps. Suitable media is poured in Petri plates
and allowed to solidify for one hour. The membrane filter is then transferred on the solidified
media in Petri plates. The plate is then incubated in inverted position 35ºC for 24hours. After
incubation the filter is dried and colonies are counted and calculated as cfu/ml of sample.

In case of no growth, the water is bacteriologically safe.

1. Pour Plate Method

This method is used separately for bacteria and fungi:

For bacteria:

10ml of sample is transferred to the Soybean Casein Digest Medium (containing

polysorbate 20) and kept at 37ºCfor 30 minutes. 1ml of solution is then pipette out to individual
sterile Petri plates. 15ml – 20ml of Soybean Casein Digest Agar is then poured in each of
above plates and incubated at 30ºC - 35ºCfor 48 – 72 hours. Plates are observed daily during
incubation.

28
At the end of incubation period, colonies cfu/ml is counted and result is reported.

For fungi:

Procedure is same as described in the test for bacteria but Sabourauds Dextrose agar
is used in placed of Soybean Casein Digest Agar and plates incubated at 22-25ºC for 5-7 days.
Colonies are counted and reported.

2. Most Probable Number

This is done to confirm whether water contains lactose fermenting gas

producing bacteria. However, it is used to determine the most probable number (MPN) of
coliforms in a sample of water besides their properties of fermenting lactose and producing gas.

In this method, three sets of four test tubes are taken. One set is taken as double
strength and two are taken as single strength. 10ml of the sample is inoculated in 10ml Double
strength MacConky’s broth tube having inverted Durham’s tubes, 1ml of sample in 5ml of
Single Strength MacConky’s broth and 0.1ml of sample in 5ml of Single Strength MacConky’s
broth. Incubation is done at 37ºC for 1-2 days. The tubes are examined for acid and gas
formation.

Confirmative test: By sub culturing on MacConky’s agar and plates are incubated at 37ºC
for 24 hours. Growth of red, non-mucoid colonies surrounded by reddish precipitate zone of
gram negative rods indicate possible presence of E.coli. This may be confirmed by sub
culturing on Eosin Methylene blue agar, incubated at 37ºC for 18-24 hours and appearance of
Metallic sheen confirm E.coli. If no such colonies appear, then E.coli is absent.

Simultaneously, control test is also carried out by streaking the culture of E.coli on
MacConkys Agar and EMB plates.

ENVIROMENTAL MONITORING

“It is a documented programme, implemented through standard operating

procedures, that describes in detail the procedures and methods used for monitoring
particulates as well as micro-organisms in controlled environment. Environmental

29
 monitoring includes sampling sites, frequency of sampling, investigative measures and
corrective measures.”

Environmental monitoring is performed to asses the aseptic processing of

bulk drug substances, dosage forms etc. it ensure the establishment, maintenance and
control of microbiological quality of controlled environments. Aseptic processing relies
on the total exclusion of micro-organisms from entering open containers during filling,
and from the process stream.

Controlled environment is defined as any area in an aseptic process system

for which air borne particulate and micro-organism levels are controlled to specific
levels, appropriate to the activities conducted within that environment.

The classification of controlled environment is based on FEDERAL

STANDARD 209E. It is a standard that “Airborne Particulates Cleanliness Classes in
Clean Rooms and Clean Zones” approved by Commissioner. Federal Supply Services,
General Service Administration, for the use of ‘All Federal Agencies”.

PURPOSE OF ENVIROMENTAL MONITORING

The main purposes of environmental monitoring are;

 It asses the effectiveness of cleaning and sanitization practices and of personnel

that could have an impact on the bio-burden of the controlled environment.

 It provides information to ascertain that the controlled environment is operating

with in an adequate state of control.

 It provides the quantization of the microbi9al content of the room air, compressor
air that enters the critical area, surfaces, equipment, sanitization, containers, floors, walls
and personnel garments.

 Environmental monitoring gives the representative estimates of the bioburden of

the environment.

 It should reflect the risks of contamination of the dosage forms to the recipient of
the pharmaceutical products.

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 RISK BASED ON ROUTE OF ADMINISTRATION

Risk of contamination is different for different products:

 Parenteral and ophthalmic products

 Inhalation solution

 Aerosol inhalants

 Nasal spray

 Vaginal and rectal suppositories

 Topical & oral liquids

In 1994, survey conducted by pharma indicated that 87% of firms conduct

microbial monitoring of air, equipments, product-contact surfaces, walls, floors and
personnel.

CLASSIFICATION OF AREA

The standard of air cleanliness is defined by the absolute concentration of air

borne particles. Federal Standard 209E as applied in pharmaceutical industry is based on
limits of all particles with sizes equal to or larger than 0.5µm.

Table:

Classes Size Limit/ft3

Class 100 0.5µm NMT 100

Class 10,000 0.5µm NMT 10,000

Class 100000 0.5µm NMT 100000

ALERT AND ACTION LEVEL:-

Alert Level:- Microbial levels, specified in the standard operating procedures, which
when exceeded should result in an investigation to ensure that the process still within
control. Alert levels are specific for a given facility and are established on the bases of a
31
 base line developed under an environmental monitoring programme. These alert levels
can be modified depending on the trend analysis done in the monitoring programme.
Alert levels are always lower than the action level.

Action Level:- Microbiological levels, in the control environment specified in the

standard operating procedure which when exceeded should trigger an investigative and
corrective action based on the investigation.

COMPENENTS:-

 Airborne non-viable particulate monitoring

 Air-borne viable contaminant monitoring

 Viable contaminant monitoring of surface

 Viable contaminant monitoring of personnel

SAMPLING LOCATION \ SITES:-

The sites selected will be based on the criticality of the operation and the sites will be
representative of locations with a potential to impact the sterility assurance of the
product.

Air monitoring should be conducted adjacent to the filling location, surface monitoring
product contact surfaces and non- contact surface adjacent to areas where product and
open vials are present while personnel monitoring should be confined to the sleeve and
gloves.

SAMPLING METHODS:-

(A). NON-VIABLE PARTICLE COUNT:-

EQUIPMENT:- METONE AIR SAMPLER

 Air is passed through a light scattering device. It then generates a signal that is
electronically processed to display particle count at different size ranges.

32
 Measures particles of sizes 0.3 µm, 0.5 µm, 1.5 µm and 10 µm.

Sampling is done according to the formulae:

N = √A

Where, N is the minimum number of sampling locations.

A is the floor area of the clean room in cubic meters.

TABLE:

CLASS PARTICLE ALERT LEVEL ACTION LEVEL

SIZE
CLASS 100 ≥ 0.5 µm 76 – 100 >100
≥ 5 µm 0.1 – 0.3 >0.3
CLASS 10000 ≥ 0.5 µm 3000 – 10000 >1000
≥ 5 µm 19 – 56 >56
CLASS 100000 ≥ 0.5 µm 50000-100000 >100000
(preparation and ≥ 5 µm 281 – 560 >560
washing room)
CLASS 100000 ≥ 0.5 µm 105-2 X 105 >200000
(other rooms) ≥ 5 µm 560 - 1000 >1000

(B). VIABLE COUNT:-

(a). ACTIVE AIR SAMPLER

(b). SETTLED PLATE TECHN IQUE OR PASSIVE AIR SAMPLING

(a). ACTIVE AIR SAMPLING:-

Equipment used:- “slit to agar air sampler”

33
 Active air samplers work on the principle of sucking or blowing a stream of air at a
sufficiently high velocity to cause the micro-organisms to be impacted against given
media.

Sampling should be done at critical zones where specific operations are being carried
out.

The unit is being powered by an attached source of controllable vacuum. The air intake
is obtained through a standardized slit below which is placed a slowly revolving Petri
dish containing a nutrient agar.

STA samplers have 80 l/min sampling capacity. If one cubic meter of air is tested, then
it would require an exposure time of 15 minutes.

(b). SETTLED PLATE TECHNIQUE:-

Container used: Petri dish

This method is widely used as a simple and inexpensive way to qualitatively asses the
environment over prolonged exposure time. The Petri plates with the agar medium are
exposed in the given area for a given time. The number of microbial particles depositing
on the plate are ascertained by the incubation followed by the counting of the number of
cfu’s.

Sampling location for settled plate count should include critical areas with little air
movement (dead space) to where air flows are excessively turbulent.

Location:-

Adjacent to doors

Supply air duct

In pass through hatches

Return air duct

34
Corners of room

Near drains

(c). SURFACE SAMPLING:-

Another component of the microbial environmental control programme in controlled

environment is surface sampling of equipments, facilities and personnel gear used in
these environments. Surface sampling is mainly done by two methods:

RODAC plate

Swab sampling

RODAC PLATE:-

RODAC stands for replicate organism detection and counting. The standard contact
plate contains sufficient agar media in such a way that it has a raised surface and can be
directly applied to test surfaces.

Location:- It is done on the equipment when the product manufactured is changed for
the new product. Sampling also depends on the critical areas and scale of operation.

SWAB SAMPLING:-

Equipment used: Dry Sterile Swab

It is done on irregular surfaces with a flat aluminum plate with a cut out hole. The area
inside is rubbed by the swab, ensuring that the complete surface is covered to recover the
maximum number of micro-organisms. The swab is then placed in an appropriate diluents
and an estimate of microbial count is done by plating an appropriate aliquot on the
specified agar medium.

Sampling is done on the irregular surface of the equipment when the product
manufactured is changed for the new product and also on the critical areas near operation.

35
Table:- Results In cfu Per Samples

CLASS LOCATIONS ALERT LEVELS ACTION LEVELS

 Critical parts 1 >1

CLASS 100 of equipment
 Other 1-3 >3
 Above floor 3-5 >5
CLASS 10000 level
 At floor level 7-10 >10
CLASS  Above floor 15- 25 >25
100000 level
(preparation  At floor level 25-50 >50
and washing)  Above floor 25-50 >50
CLASS level
100000  At floor level 50-100 >100
(other areas)

Mainly two methods are employed for personnel monitoring:

Finger print

RODAC on gowning

FINGER PRINT: - With gloves on, the finger tips are pressed onto the Petri plates
containing solidified medium. The gloves are discarded immediately thereafter and the
plates are incubated to ascertain the number of cfu’s.

RODAC ON GOWNING:- This is done on the worker’s gowns on the lower

ends of the, chest and the collar, in the morning towards recess and at the end of the shift.

36
TABLE: 3

LOCATION ALERT LEVEL ACTION LEVEL

Persons operating in 2-3/3-5 >3/>5

preparation and filling
rooms

Persons in green area 5-10 >10

Culture Media and Diluents Used for Sampling and Quantization of b

Micro- Organisms:-

The type of medium, liquids or solids that is used for sampling or quantization of micro-
organisms in controlled environments will depend on the procedures and equipments
used. A commonly used all purpose medium is Soybean Casein Digest Agar when a solid
medium is needed. Other media, liquids or solids are as follows:

Table:

LIQUID MEDIA SOLID MEDIA

o Tryptone saline Soybean casein digest agar

o Peptone water Nutrient agar

o Buffered saline Lecithin agar

o Buffered gelatin Tryptone glucose extract agar

o Brain heart infusion

o Soybean casein medium

37
 Table:

SOLID DOSAGE
TESTS AREA MICROBIOLOGY
( 90 locations)

o Non-viable count Every 3 month Daily

o Settled plate count 15 days Daily

o Active air sampler 15 days Daily

o RODAC/SWAB During cleaning validation Daily

o Finger printing and

Weekly Daily
RODAC on gowning

CONCLUSION

38
 A well designed and executed environment monitoring is an
essential QA activity for the manufacture of products.

 A periodic environmental monitoring programme should be

established for high risk non-sterile products.

 Programme must be designed in a manner defendable during

compliance audit.

REFERENCES

39
 Blechova R., D Pivodva; LAL Test – a alternative method of dilution of bacterial

endotoxin Acta Vet. Brno 2001; 70; 291- 296.

 Cooper JF, Rounding BET related calculations. LAL. Times 5: 3, 1998.
 Danyer S.P and Hodges (NA) (1989) “Theory and Application of Microbiological

Assay” Academic Press London.

 http:\\en-wikipedia. org/wiki/limulus amehocyte lysate.
 Indian Pharmacopoeia (1999) “Method for Antibiotic Assay.”
 M. Jezowska-Bojczuk, “Structure of gentamicin” (1998).
 Prescott, H. Kelin; Microbiology (6th edition) 2005 (History of Antibiotics)
 United State pharmacopoeia – 28(2005) “Method for antibiotic Assay”.
 United state pharmacopoeia – 26 (2006) II (2513-2526)
 United state pharmacopoeia – 28 (2008) “Quality of Antibiotics”

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