"THE IMPORTANCE AND APPLICATION OF BACTERIAL

ENDOTOXIN TEST IN PHARMACEUTICAL INDUSTRY".

An INTERNSHIP REPORT
SUBMITTED BY

Bhuva Trutik R.
Enrollment No: 2156003008

As a partial fulfillment of Internship for the award of the degree

of

MASTER OF SCIENCE
IN
BIOTECHNOLOGY

(Internal Guide) (External Guide)

Ms. Drashti mam Dr. Mitesh Patel

Assistance professor, Technical Director
Department of of QC Microbiology
Biotechnology, and Fermentation
Department at
Sumar biotech LLP.

Swarrnim Startup & Innovation University

JANUARY 2023
 CERTIFICATE

This is to certified that the work presented in the project report entitled “THE
IMPORTANCE AND APPLICATION OF BACTERIAL ENDOTOXIN TEST
IN PHARMACEUTICAL INDUSTRY, SAFAL LIFE SCIENCE Pvt. Ltd’’
submitted by Bhuva Trutik R. Enrollment No: - 2156003008 of Biotechnology
department of the Swarrnim Startup & Innovation University comprises the work carried
out under my supervision for the partial fulfilment of the award of the degree of M.Sc.
Biotechnology of the Swarrnim Startup & Innovation University.

Ms. Darshti mam Ms. Archana pandey

Assistant professor, Department of Head of the Department,
Biotechnology, Swarrnim Startup & Biotechnology, Swarrnim
Innovation University Startup & Innovation University

Date: - / /2023
 LIST OF ABBREVIATION

QA: Quality Assurance

QC: Quality Control

SOP: standard Operating procedure

LAL: Limulus Amebocyte Lysate

LAF: Laminar Air Flow

WFI: Water for Injection

MVD: Maximum Valid Dilution

CSE: Control Standard Endotoxin

BET: Bacterial Endotoxin Test

LPS: Lipopolysaccharide

EU: Endotoxin Unit

WBC: White Blood Cell

USP: USA Pharmacopoeia

CFG: Caspofungin

NC: Negative Control

PC: Positive Control

PPC: Product Positive Control

NPC: Product Negative Control

GM: Geometric Mean

 ACKNOWLEDGEMENT

The internship opportunity I had with Sumar Biotech LLP, was a great chance for learning
and professional development. Therefore, I consider myself lucky to be a part of it. I also
had a great opportunity to learn and work with professionals who guided Dr Mitesh Patel
internship period.

Bearing this in mind, I take this opportunity to express my deep sense of gratitude and
heartly thanks to My guide, Dr. Mitesh Patel Technical Director of Fermentation and QC
[microbiology department] who have guided throughout the internship and encouraged to
develop interest in the field, in spite of being busy with routine work. She also has inspired
me to develop a positive attitude towards work and develop good communication skills
and its importance for working in a company.

My deepest gratitude to Ms. Trupti Patel (HR Manager) to provide proper facilities in the
company premises which made it easier for me to carry out internship. My sincere
gratitude to Ms. Dashanki Pancholi, Ms. Krupali Patel and Ms. Avani Patel for
imparting knowledge, kind help, advice and support during the internship.

I will express my deepest thanks to Ms. Drashti for giving necessary advice and guidance
as and when needed, I will choose this moment to acknowledge her contributions fully.

I will also like to thank Ms. Archna pandey Head of Biotechnology Department also
extend my thanks to entire faculty members of the department who had actively or
passively helped me during my dissertation work.

I want to express my thank to my beloved parents and family members for their love,
support and good wishes I would like to extend my thanks to all my friends and
batchmates for their precious support
 INDEX

ABSTRACT

CHAPTER 1 INTRODUCTION 01

CHAPTER 2 REVIEW OF LITERATURE 07

MATERIALS
CHAPTER 3 13
&METHODS

CHAPTER 4 RESULTS AND DISCUSSION 25

CHAPTER 5 CONCLUSION 29

CHAPTER 6 REGERENCES 31

.
 Abstract

The Bacterial endotoxin testing is essential part of every injectable pharmaceutical industry of finished

product that are used to treatment of various diseases. Therefore, testing in laboratory is must be

required and out of various test bacterial endotoxin test is one of the important tests ensure about that

the product which is completely sterile and free from microbial contamination or load and free from

pathogen. Bacterial endotoxin test is quantifying and detect endotoxin from gram negative bacteria

using amebocyte lysate extracted from Limulus Polyphemus horseshoe crab blood is used to prepare

lysate there are three techniques are used for the detect of the endotoxins in over finished product.

1) gel-clot technique is based on the gel formation.

2) turbidimetric technique is depends upon the development of turbidity after the destruction of

substrate.

3) chromogenic technique is based on the formation of color after the cleavage of synthetic peptide

chromogen complex, endotoxins are toxic complexes which is associated with the lipopolysaccharide

outer membrane of cell wall of the gram negative bacteria pathogenicity pyrogen are known as

endotoxin carrying bacteria they cause high fever when its enter the patient body the test can be done

by these three techniques but most of the use in pharmaceutical industry is gel-clot technique require

the incubation period is 1 hours at 37c temperature the test is carried out in a avoiding endotoxins

contamination mostly injectable pharmaceutical industry bacterial endotoxin test .Endotoxin had been

high fever contain thus are very necessary removed out from any type of microbial contamination out

from the directly use injectable pharmaceutical product.

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CHAPTER 1

INTRODUCTION

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INTRODUCTION

Endotoxins are toxic complexes which is invariably associated with the cell-wall of the Gram-negative

bacteria irrespective of bacterial pathogenicity. Endotoxins are rarely fatal, but they cause fever and

hence Endotoxin carrying bacteria are known as Pyrogen. Endotoxin is toxic substances which is bound

with the bacterial cell wall and are released when the bacterium disintegrates. Endotoxin composed of

lipopolysaccharide and lipoprotein complexes.

The components that are protein determine its antigenic property; and the polysaccharide component

determines the antibody type that can react with the Endotoxin molecule. The reaction of

polysaccharide components with the Endotoxin when allowed produces an immune reaction. Endotoxin

is rarely fatal, although they often cause fever. Thus, Bacterial Endotoxin test is the confirmatory test

that assures the presence or absence of the Endotoxin in the medicine sample. Mostly bacterial

Endotoxin test are performed for the inject-able pharmaceutical products, e.g. Caspofungin and other

Micafungin, Anidulafungin and Glutathione All these products have their specific Endotoxin limit

value determined by EU/mg.

These bacteria are characterized by their cell envelopes. Their cell wall is composed of a thin

peptidoglycan cell-wall sandwiched between an inner cytoplasmic cell membrane and a bacterial outer

membrane. Gram-negative bacteria are found almost everywhere. The gram-negative bacteria include

the model organism Escherichia coli, as well as many pathogenic bacteria. Their outer membrane

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protects them from many antibiotics (including penicillin); detergents that would normally damage the

peptidoglycans of the (inner) cell membrane; and lysozyme, an antimicrobial enzyme produced by

animals that forms part of the innate immune system. Additionally, the outer leaflet of this membrane

comprises a complex lipopolysaccharide (LPS) whose lipid A component can cause a toxic reaction

when these bacteria are lysed by immune cells. This toxic reaction can include fever, an increased

respiratory rate, and low blood pressure a life-threatening condition known as septic shock

LPS is pyrogenic, and bacterial endotoxin is a synonym for LPS. LPS is the toxin which is synthesized

endogenous to the bacteria cell structure. When Gram-negative bacteria are destroyed, endotoxin is

released. In the human body, endotoxin triggers the activation of the body’s defense system, which, in

turn, elevates the body temperature and elicits the pyrogenic response. LPS is located outside a thin

structural layer of peptidoglycan.

GRAM-NEGATIVE BACTERIA:

In the method of bacterial differentiation such bacteria do not retain the crystal violet stain used in the

gram-staining.

These bacteria are characterized by their cell envelopes. Their cell wall is composed of a thin

peptidoglycan cell-wall sandwiched between an inner cytoplasmic cell membrane and a bacterial outer

membrane. Gram-negative bacteria are found almost everywhere. The gram-negative bacteria include

the model organism Escherichia coli, as well as many pathogenic bacteria. Their outer membrane

protects them from many antibiotics (including penicillin); detergents that would normally damage the

peptidoglycans of the (inner) cell membrane; and lysozyme, an antimicrobial enzyme produced by

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animals that forms part of the innate immune system. Additionally, the outer leaflet of this membrane

comprises a complex lipopolysaccharide (LPS) whose ‘lipid A’ component can cause a toxic reaction

when these bacteria are lysed by immune cells. This toxic reaction can include fever, an increased

respiratory rate, and low blood pressure a life-threatening condition known as septic shock. Some

examples of Gram-negative bacteria that have demonstrated drug resistance include.

ENDOTOXINS: -

The bacteria those are associated with Endotoxin grows side by side with fungus and or mold in watery

environments specifically. These gram-negative micro-organisms (bacteria) collectively get

accumulated in the human digestive system and thus these microorganisms are responsible for the

inflammation in the gut and also in different parts of the body. Endotoxin had been authenticated as

inflammatory agent, onset clinical diabetes, obesity, nausea, vomiting, diarrhea, fever, disseminated

intravascular coagulation, vascular collapse, and organ failure. Endotoxins are thus very necessary to

get removed eliminated out from the inject-able pharmaceutical products.

AMOEBOCYTE LYSATE

The term lysate is defined as the mixture of substances which is formed by the lysis of the cells.

Amebocyte lysate is associated with the WBC of horseshoe crab (Limulus polyphemus or Tachypleus

tridentatus). Amebocyte lysate is a lyophilized product procured from the lysate of amebocyte from the

horseshoe crab. Lysate those are obtained from Limulus polyphemus is known as Limulus Amebocyte

Lysate, while lysate those are obtained from Tachypleus tridentatus is known as Tachypleus

Amebocyte Lysate. Amebocyte lysate is needed to be dissolved in the water associated

with the types of lysate e.g. for Limulus Amebocyte Lysate the water that is needed to reconstitute the
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lysate is thus LAL Reagent Water whereas for Tachypleus Amebocyte Lysate the water that is needed

to reconstitute the lysate is thus TAL Reagent Water. Lysate is lyophilized and it should be stored at 2-

8 ◦ C.

AIM:

To Perform Bacterial endotoxin test for Caspofungin.

To Perform Bacterial endotoxin test for Micafungin.

To Compared and Evaluation the Bacterial endotoxin test for different finished product.

OBJECTIVE:

Endotoxins from gram-negative bacteria are the most common cause of toxic reactions resulting from

the contamination of pharmaceutical products with pyrogen. This endotoxin if present in the material

under test reacts with the lysate to form turbidity, precipitate or gel. This procedure describes the

method to be followed for analyzing bacterial endotoxin.

1. Distinguish the requirements for BET test.

2. Understand the basic principles and procedure for BET test.

3. Observe, report, and interpret the results of BET test.

4. Develop corrective action(s) for out-of-specification finding(s) of BET test.

5) Identify the sources of bacterial endotoxin in the preparation of injectable radiopharmaceuticals

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such as an F-18 product.

6) Describe the reagents, materials and equipment needed for the BET.

7)Understand how to select and establish a BET method.

8)Define volumes, solutions and containers needed to prepare a radiopharmaceutical for a BET.

9)Explain the calculations for the endotoxin limit and limit of detection (LOD).

10). Identify the advantages of the photometric-BET in contrast with the gel-clot BET.

11) Describe the procedure for qualifying a LAL reagent and an analyst for the BET.

12). Identify the principle cause of invalidity in a photometric or gel-clot BET.

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CHAPTER 2
REVIEW OF LITERATURE

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REVIEW OF LITERATURE

Sterile products are parenteral products which are injected through the skin or mucous membranes into

internal body components. They should be free from microbial contamination, from toxic components

as well as should be sterile. All the processes involved must be performed under standard operating

procedures and designed to eliminate contamination of all types whether physical, chemical or

microbiological origin. Pyrogens are produced mainly by gram negative bacteria and form a part of

endotoxin (osmotic antigen). Many medicinal agents if present interfere with the test results because of

their antipyretic or other interfering effects. Hence pyrogen test is performed on those finished products

which do not interfere with the test. Drugs that are to be intravenously injected must be of a pyrogen-

free quality. For other parenteral drugs, given subcutaneously or intramuscularly in much smaller

volumes (e.g. Vaccines) a maximal acceptable endotoxin concentration has to be defined for quality

control purposes. (Mulay Sushruta*, 2011).

According to the Khale Anubha, water is used as the vehicle for most injections as aqueous

preparations are tolerated well by the body and are safest and easiest to administer. In preparation of

water for injection removal of entrained contaminants from the vapor before it is condensed by passage

through an efficient baffle system eliminate pyrogens.

According to the USP chapter <85>Bacterial Endotoxin Test, the Gel-clot techniques are used to

detect and quantify the bacterial endotoxin based on clotting of LAL reagent in the presence of

endotoxin. The concentration of endotoxin required to cause the lysate to clot under well-defined

conditions is the labelled sensitivity of LAL reagent.

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According to the USP chapter <85>Bacterial Endotoxin Test, there are two types of techniques for this

test: the gel-clot techniques, which are based on gel formation, and the photometric techniques. The

latter include a turbid metric method, which is based on the development of turbidity after cleavage of

an endogenous substrate, and a chromogenic method, which is based on the development of color after

cleavage of a synthetic peptide chromogen complex. Proceed by any one of these techniques, unless

otherwise indicated in the monograph. In case of dispute, the final decision is based on the gel-clot

techniques, unless otherwise indicated in the monograph. In the gel-clot techniques, the reaction

endpoint is determined from dilutions of the material under test in direct comparison with parallel

dilutions of a reference endotoxin, and quantities of endotoxin are expressed in USP Endotoxin Units

(USP-EU).

Bacterial endotoxin is the lipopolysaccharide (LPS) component of the cell wall of Gram-negative

bacteria. It is pyrogenic, and it is a risk to patients who are administered intravenous and intramuscular

preparations. Pyrogenicity and the structure of endotoxin and moves onto examine Limulus amebocyte

lysate (LAL) testing and other, alternative, methods of assessing pyrogens and endotoxin. For these

reasons, pharmaceutical products that are injected into the human body are tested for pyrogenic

substances. The most common, and arguably most important pyrogen, is bacterial endotoxin. Bacteria

endotoxin presents a significant risk to many pharmaceutical products, especially parenteral products.

According to World health organization, the gel-clot technique is for detecting or quantifying

endotoxins based on clotting of the lysate TS in the presence of endotoxin. The minimum concentration

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of endotoxin required to cause the lysate to clot under standard conditions is the labeled sensitivity of

the lysate TS. To ensure both the precision and validity of the test, perform the tests for confirming the

labeled lysate sensitivity and for interfering factors as described below under Preparatory testing.

This technique is a photometric assay measuring increases in reactant turbidity. On the basis of the

particular assay principle employed, this technique may be classified as either an endpoint-turbidimetric

assay or a kinetic-turbidimetric assay. The endpoint-turbidimetric assay is based on the quantitative

relationship between the concentration of endotoxins and the turbidity (absorbance or transmission) of

the reaction mixture at the end of an incubation period. The kinetic-turbidimetric assay is a method to

measure either the time (onset time) needed to reach a predetermined absorbance or transmission of the

reaction mixture, or the rate of turbidity development. The test is carried out at the incubation

temperature recommended by the lysate manufacturer (which is usually 37 ± 1 °C).

This technique is an assay to measure the chromophore released from a suitable chromogenic peptide

by the reaction of endotoxins with lysate. On the basis of the particular assay principle employed, this

technique may be classified as either an endpoint-chromogenic assay or a kinetic-chromogenic assay.

The endpoint-chromogenic assay is based on the quantitative relationship between the concentration of

endotoxins and the release of chromophore at the end of an incubation period. The kinetic-chromogenic

assay is a method to measure either the time (onset time) needed to reach a predetermined absorbance

of the reaction mixture, or the rate of color development. The test is carried out at the incubation

temperature recommended by the lysate manufacturer (which is usually 37 ± 1 °C).

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The rabbit test is no longer widely used, and it has largely been replaced, for the testing of parenteral

drug products, by the LAL test. The reason for this is because the most common type of pyrogen found

in the pharmaceutical industry is bacterial endotoxin, and for which, LAL (with some limitations

explored below) is a specific test for. This risk from endotoxin is due, not least, to the large quantities

of water used in the manufacture of pharmaceutical products as Chapter 10 describes. The

predominance of the LAL assay is not to suggest that rabbit pyrogen testing has been completely

eliminated, but that its use is in decline. Moreover, there are alternatives to the LAL test, such as

enzyme-linked immunosorbent assay (ELISA) methods and the monocyte activation test (MAT).

The LAL test is a method, of the bacterial endotoxin test (BET), for detecting the presence, and to go

some way to determining the level, of Gram-negative bacterial endotoxins in a given sample or

substance. Current editions of the pharmacopoeia carry statements to the effect that where the term a

pyrogenic or pyrogen-free is used it should be interpreted as meaning that samples of the product will

comply with a limit for bacterial endotoxin. However, it was not until the early twentieth century with

the development of a rabbit pyrogen test that an understanding emerged in which bacteria could be

classified into pyrogenic and nonpyrogenic types, creatable to their Gram stain. Gram- negative

bacteria were found to be pyrogenic, Gram-positive bacteria were generally not: and killed cultures of

Gram-negative bacteria were comparable to live cultures in their ability to induce fevers. Due to the

association with living and dead bacteria, by the 1920s, it was apparent that sterility in parenteral

pharmaceuticals could be no guarantee of nonpyrogenicity, and that if pyrogenicity was to be avoided it

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was imperative to avoid bacterial contamination at every stage of manufacture of parenteral

pharmaceuticals.

The structural rigidity of the bacterial cell wall is conferred by a material called peptidoglycan (also

known as murein). It is a polymer consisting of sugars and amino acids that forms a mesh-like layer

outside the plasma membrane of bacteria forming the cell wall. Thin layer, and it is not the outermost

layer. Gram-negative bacteria instead have an outer membrane, and they are sometimes described as

having a cell envelope rather than a cell wall. The outer membrane functions to maximize the ability of

the bacterium to derive nutrients from the external environment. The outer layer also functions as a

permeability barrier effective against diffusion of exo-enzymes into the external environment. This is

an evolutionary feature that has arisen to allow Gram-negative bacteria to survive and increase in

numbers in environments such as water in which there are only low concentrations of organic nutrients.

Macromolecular organic nutrients are trapped in the cell envelope as the water flows by, and then

within the cell envelope they are hydrolyzed to smaller molecules that can be taken into the cell.

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Chapter:3
Material and Methodology

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Equipment required:

1) vortex mixture.

2) Heating block.

3) Micropipette (1000microliter/200 microliter).

4) oven.

5) pH meter.

6) Thermometer.

7) Refrigerator.

8) Hot air oven for depyrogenation.

Material required:

Limulus amebocyte lysate [LAL] with sensitivity of 0.03 EU / ml, Reference Standard Endotoxin or

Controlled standard endotoxin, Depyrogenated Glass pipette, Test tubes with

S.S. caps (38 x 200 mm), Dilution Tubes (12x75 mm), Incubation tubes (10 x 75 mm), measuring

cylinder, Micropipette and tips.

1) Caspofungin (5EU/ml).
2) Micafungin (0.6EU/ml).

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Utilities required:

Water for injection, electricity

Methodology for bacterial endotoxin test:

The structural rigidity of the bacterial cell wall is conferred by a material called peptidoglycan (also

known as murein). It is a polymer consisting of sugars and amino acids that forms a mesh-like layer

outside the plasma membrane of bacteria forming the cell wall [8]. In Gram-positive bacteria,

peptidoglycan is present as a thick layer that is outer-most in the cell wall. In Gram-negative bacteria,

the peptidoglycan is only a thin layer, and it is not the outermost layer. Gram-negative bacteria instead

have an outer membrane, and they are sometimes described as having a cell envelope rather than a cell

wall. The outer membrane functions to maximize the ability of the bacterium to derive nutrients from

the external environment. The outer layer also functions as a permeability barrier effective against

diffusion of exo-enzymes into the external environment. This is an evolutionary feature that has arisen

to allow Gram-negative bacteria (illustrated in Figure 11.1) to survive and increase in numbers in

environments such as water in which there are only low concentrations of organic nutrients.

Macromolecular organic nutrients are trapped in the cell poison as the water flows by, and then within

the cell envelope they are hydrolyzed to smaller molecules that can be taken into the cell.

Bacterial endotoxin test is used to quantify or detect endotoxins from Gram negative bacteria using

amebocyte lysate from horseshoe crab. Endotoxin: lipopolysaccharide associated with outer

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membrane of gram-negative bacteria such as pathogens such as Escherichia coli, Salmonella, Shigella,

Pseudomonas, Neisseria, Hemophilus influenzae, Bordetella pertussisand Vibrio cholerae.

There are three methodology use in pharmaceutical industry

1) Gel - clot method

2) Turbidimetric method

3) Chromogenic method

1) Gel - clot method

Depyrogenation: Depyrogenate the glassware by heating at 250 t 10 ° C for 1 hour in hot air oven and

treat micro tips with 4% NaOH solution for Depyrogenation (follow SOP SL / QC / 31 for

depyrogenation). Use the glassware and WFI after cooling to room temperature.

Reconstitution of Endotoxin:

Reconstitute the endotoxin as per the label instruction on the vial, using freshly collected water for

injection. Vortex the endotoxin vial at least for 30 minutes vigorously on vortex mixture at high speed.

Prepare the endotoxin dilution equivalent to labeled sensitivity 0), twice of labeled sensitivity (22), four

times of labeled sensitivity (4) and half of the labeled sensitivity (/ 2) of the lysate used using freshly

collected water for injection.

Vortex at each step during dilution for 2-3 minutes.

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Lysate reconstitution:

Before using the lysate ensure that it has been confirmed / validated for its claimed sensitivity.

Bring the lysate from refrigerator and reconstitute it with water for injection as per labeled instruction.

Swirl it gently between palms for 1-2 minutes. Take care not to produce foam / froth in the vial.

Dilution of the sample to be tested:

Prepare half the dilution (i.e. MVD) of materials using freshly collected water for injection in

Depyrogenated test tubes. Label the tubes appropriately.

Check the pH. Adjust it between 6.00 to 8.00 if required by using Pyrogen free 0.1M NaOH or 0.01 M

NAOH or by using of 0.1 M HCI solution.

Label the 10 x 75 mm tubes appropriately and arrange them in stand.

Perform the tests for negative control (WFI), positive control, sample and positive product control in

duplicate.

With the help of micropipette take each of negative control, positive control, product positive control

and sample as mentioned in Table- 1.

Add 0.1 ml of Lysate to each tube. Shake the tubes gently for a couple of seconds and incubate the

tubes in the heating block, previously adjusted at 37.0 t 1.0 ° C for 60.0 +2.0 minutes (Ref: BP / Ph.

Eur).

Note: Performed the entire test under the Laminar airflow bench. Switch "ON" the Laminar air flow

bench at least one hour before the test Performed and continue till completion of the test observation.
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Expose the settle plate in the LAF bench during the analysis for the checking of microbial

contamination. (Frequency: once in a month)

Table-1

Sr. No. TUBE WFI SAMPLE ENDOTOXIN LYSATE

Negative
1. 0.1 - - 0.1 ml
control
Positive
2. - - 0.1 ml (2λ) 0.1 ml
control
Positive
3. product - 0.05 ml 0.05 ml (4λ) 0.1 ml
control
Specimen
4. - 0.1 ml - 0.1 ml
(MVD)

After incubation observe the tubes by slowly inverting the tube upside down at 180 ° angle to check the
formation of gel.

Table-2

Product
Negative Positive Action to be
Case Specimen positive
control control taken
control

Sample passes
1. -ve -ve +ve +ve -ve -ve +ve +ve
the test

Repeat the test

by using double
2. -ve -ve +ve +ve -ve +ve +ve +ve
the number of
samples.
Repeat the test
by using double
3. -ve -ve +ve +ve +ve +ve +ve +ve
the number of
samples.
Repeat the
4. -ve -ve +ve +ve -ve -ve -ve -ve using MVD if
not used.

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Repeat the test
5. -ve -ve -ve +ve -ve -ve -ve -ve with fresh
LAL/Endotoxin
Repeat the test
6. +ve +ve +ve +ve +ve +ve +ve +ve with fresh
LRW or WFI

7. -ve +ve -ve +ve -ve +ve -ve +ve Repeat the test

Methodology for confirmation of lysate sensitivity:

Depyrogenation:

Depyrogenate the glassware and NAOH pellets by heating at 250 ° C + 10 ° C for 1 hours in hot air

oven and microtips treat with 4% NaOH solution for depyrogenation (follow SL / QC / 30 for

depyrogenation). Use the glassware and WFI after cooling to room temperature.

Reconstitution of Endotoxin:

Reconstitute the endotoxin as per the label instruction on the vial, using freshly collected pyrogen free

water for injection / LAL reagent water.

Vortex the endotoxin vial, at least for 30 minutes, on vortex mixture at high speed.

Prepare the dilution as follows Reconstitute Control Standard Endotoxin (CSE) as per manufacturers

instruction. Prepare a 4-replicate series of 2-fold dilution of the controlled standard endotoxin to make

concentration of 22, 2, 2, 4. Dilute it as follows to get 1 EU/ml concentration and them serially till

0.015 EU / ml concentrations.

0.1 ml CSE+{0.1×(X-1) ml of WFI = 1.0 EU/ ml (a)

1.0 ml (a) + 1.0 ml WFI = 0.5 EU/ml (b)

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1.0 ml (b) + 1.0 ml WFI = 0.25 EU/ml (c)

1.0 ml (c) + 1.0 ml WFI = 0.125 EU/ml (d)

1.0 ml (d) + 1.0 ml WFI = 0.06 EU/ml (e)

1.0 ml (e) + 1.0 ml WFI = 0.03 EU/ml (f)

1.0 ml (f) + 1.0 ml WFI = 0.015 EU/ml (g)

X=Endotoxin concentration EU/ml

Carry out BET using these dilutions of endotoxin as per method described in 4.4. Observe the results

after completion of incubation period.

a) If the gel that remains intact momentarily when tube is inverted, the result is positive.

b) If the tube content is semisolid or liquid the result is negative.

Calculate the geometric mean: The end point is the last positive test in a series of decreasing

concentration of endotoxin. Record the end point concentration for each replicate series of dilution.

Determine the log endpoint concentration and calculate the geometric mean end point concentration

(GM) using the following formula.

GM - Antilog (Ee / f)

Where Ee = Sum of log end point concentration

F= No. of replicates

Record the results in confirmation of the lysate sensitivity test protocol (Annexure 3).
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Methodology for test of interfering factor (Inhibition/Enhancement):

Depyrogenation:

Depyrogenate the glassware by heating at 250 ° C + 10 ° C for 1 in hot air oven. Use the glassware and

water for injection after cooling to room temperature.

Reconstitution of Endotoxin & testing:

Reconstitute the endotoxin as per the label instruction on the vial, using freshly Vortex the endotoxin

vial, at least for 30 minutes, on vortex mixture at high speed. Prepare a 4-replicate series of 2-fold

dilution of the controlled standard endotoxin to make concentration of 2., A, N2, / 4. Prepare the

dilution as follows:

a. Reconstitute CSE as per manufacturer's instruction. Dilute it as follows to get 1 EU/ ml concentration

and then serially till 0.015 EU/ ml concentrations.

0.1 ml CSE+{0.1×(X-1) ml of WFI = 1.0 EU/ ml (a)

1.0 ml (a) + 1.0 ml WFI = 0.5 EU/ml (b)

1.0 ml (b) + 1.0 ml WFI = 0.25 EU/ml (c)

1.0 ml (c) + 1.0 ml WFI = 0.125 EU/ml (d)

1.0 ml (d) + 1.0 ml WFI = 0.06 EU/ml (e)

1.0 ml (e) + 1.0 ml WFI = 0.03 EU/ml (f)

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1.0 ml (f) + 1.0 ml WFI = 0.015 EU/ml (g)

X=Endotoxin concentration EU/ml

Perform the test on aliquots of the specimen or a dilution not to exceed the MVD.

Carry out BET using above dilution and controls as per method described in 4.04.

Calculate the geometric mean.

2) Turbid metric method:

The turbid metric assay is, together with the chromogenic assay, a photometric method. With this test,

during the process of clot formation, the lysate-sample reaction mixture becomes increasingly more

turbid. During the LAL reaction, the concentration of insoluble coagulin increases as its precursor,

coagulogen, is cleaved. This process causes a corresponding increase in optical density (OD) of the

reaction mixture. It is this increase in OD that is detected in a spectrophotometer (typically a

microplate reader or a tube reader). The rate of increase of the OD is directly proportional to the

endotoxin concentration present in the well. This is the basis for the turbid metric LAL assay. The

assay measures the increase in turbidity as a function of endotoxin concentration measured against a

standard curve and, from this, estimates the endotoxin concentration in a sample.

3) Chromogenic method:

The chromogenic assay uses the initial portion of the endotoxin cascade. Here a synthetic chromogenic

peptide is substituted for the clotting protein the peptide generates a yellow color. The chromogenic

assay uses a synthetic chromogenic substrate that contains a specific sequence of amino acids that are

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designed to mimic the cleavage site in co-agulogen. Activated clotting enzymes cleave this site and

cause the liberation of the chromophore (para-nitro aniline. PNA), which has a yellow color. The

liberated PNA absorbs light at 405 nm. In the chromogenic assay, the measurement of this absorption

of light at this 405 nm that is measured. The degree and the rate of increased absorption is proportional

to the endotoxin within the sample. When using kinetic methods, the most important aspect is the

standard curve for the endotoxin concentrations. This is because the standard curve selected, and how

it performs, determines the test sensitivity. Therefore. the high point and low point in a standard curve

determine the lower and upper levels of endotoxin that can be detected Although the LAL test today is

more robust, it remains open to a degree of variation.

Limulus Amebocytes lysate test application:

As indicated above, the widest application of the LAL test method is with the testing of samples of

water (primarily water-for-injection; WFI) and for assessing final products, especially those

administered by injection. A related area is with depyrogenation studies. WFI and sample testing are

important because endotoxicity is not necessarily lost with the loss of viability of micro-organisms.

LPS is not destroyed to any significant extent by sterilization treatments such as steam sterilization,

gamma radiation. ethylene oxide, and hydrogen peroxide. LPS also passes through 0.22-um bacteria-

retentive filters. It is claimed that endotoxin may be removed from liquids by up to 4log reductions

using 0.025-pm ultrafilters (which function as a molecular sieving process).

With depyrogenation, there are two commonly used ways of eliminating endotoxin from materials,

removing them and inactivating them: by rinsing or by dry heat depyrogenation. The normal method of

removal is by rinsing the material with WFI. This is normally applied to rubber stoppers for vials. It is

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also what is done to vessels and major pieces of equipment used for sterile parenteral manufacturing. A

question arises as to whether this can be validated and assured. In answer, the sampling statistics is

likely to be poor, and the test method is inaccurate and probably there is not much there in the first

place.

Inactivation is achieved by dry heat. If there are materials and glass vials that are required for sterile

parenteral manufacturing that can be depyrogenated by dry heat, then they should be depyrogenated by

dry heat in an oven or a tunnel. The regulatory standard for validation of an endotoxin inactivation

(depyrogenation) process is that it should be capable of reducing an endotoxin challenge through 3 log,

reduction. To ensure that this limit works there is also a requirement to clean materials prior to dry heat

depyrogenation with WFI-otherwise at least in theory, an item could be contaminated with 16,000 EU

prior to entering a validated endotoxin inactivation process and still emerging with 10 EU intact and

ready to contaminate the product.

Dry heat depyrogenation is a complex process that is still poorly understood with contradictory

research data. The phenomenon that complicates the picture is that in- activation may approximate to

the second-order chemical kinetics with a high initial rate of inactivation, then tail off to nothing. What

this means in practice is that, at any particular depyrogenating temperature, it will be subject to some

degree of inactivation in some period of time or other but beyond that point no further inactivation will

occur by holding the material at that temperature.

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Chapter 4
RESULT AND DISCUSSION

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4.1 RESULTS OF BET:

As for example,

Let’s take Sample Caspofungin, and its Endotoxin Limit: 5 EU/mL,

Lysate Sensitivity (λ): 0.03 EU/Ml.

Then, MVD calculation will be,

MVD = 5 x 10
0.03 EU/mL

= 1:1666.6=1:1600

[Routine dilution is MVD; hence sample needs to be diluted till MVD/2 to do testing on 50:50
dilution method]
Therefore,
MVD/4 =
1:400

Sample Dilution:
Take 1ml of sample A and dilute it with 1mL LRW (MVD/4)

Control Standard Endotoxin (CSE) Dilution:

20 EU/mL --- 0.1 mL CSE + 7.9 mL LRW (4λ).

Dilution Preparation:
Dilution for LRW Sample CSE Lysate

Negative Control 100 µL - - 100 µL

Positive Control 50 µL - 50 µL 100 µL

Product Positive
- 50 µL 50 µL 100 µL
Control
Negative Product
50 µL 50 µL - 100 µL
Control (Sample)

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Incubation Time: 60 min ± 2 min.
Incubation Temperature:37°C±1°C

Results:

Dilution for Tube -1 Tube - 2

Negative Control Negative Negative

Positive Control Positive Positive

Product Positive Control Positive Positive

Negative Product Control
(Sample) Negative Negative

Legend: Negative means no gel firm after inverting tube at 180° and
Positive means gel form after inverting tube at 180°
Observation:

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 NC should be negative i.e. no gel clot formation when the tubes are inverted 180°

 No gel clot formation in NPC indicates that the sample is passing the test

 Formation of firm gel in NPC indicates that the sample is failing the test

 PC and PPC should be positive i.e., formation of firm gel capable of maintaining its

integrity when the tube is inverted 180°

 Formation of firm gel in PPC indicates that the product has no interference with the test

Test consider as invalid if following observation are found:

 If any positive control / product positive control tubes are negative.

 If any negative control tube is positive.

 If negative product control one tube gives positive reaction and second tubes gives

negative reaction.

Observation: The sample contains less than 5 EU/mL of endotoxin.

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Chapter 5
Conclusion

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CONCLUSION

Conclusion The substance / preparation complies with the bacterial endotoxin test if product

positive control is positive and negative controls are negative. If positive results are obtained for

one of the test duplicates, then repeat the test once again. If samples show negative results the

product / preparation complies with the test and it fails / doesn't comply if either one or both of the

duplicates show gel formation.

Pyrogens are a concern for pharmaceutical rue products and for many of the ingredients used to

formulate them. This is especially so for products that come into contact with human blood. Here,

by far the most concerning pyrogen is bacterial endotoxin. In relation to this, the chapter has

considered the risks of endotoxin to pharmaceutical processing and some of the control measures

in place to reduce the risk of contamination. Furthermore, the chapter has provided an introduction

to endotoxin as well as to the primary method for detecting endotoxin: the LAL test. Here the

chapter has examined the three main types of LAL test gel-clot, chromogenic, and turbidimetric, as

well as considering alternative test methods. Such tests are an essential feature of most

pharmaceutical microbiology laboratories. The Limulus Amebocyte Lysate reacts with endotoxin

due to cascade of enzymatic reaction resulting in formation of gel. This reaction depends on

concentration of the endotoxin. The lysate sensitivity refers to the minimum concentration that can

be detected by using the lysate. Each batch of lysate obtained from the supplier has to be

confirmed for its claimed labeled sensitivity. Acceptable variation is one half to two times the

labeled sensitivity i.e. N2 to 2. The lysate is therefore said to be validated to the claimed

sensitivity if the calculations give variation in above range.

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CHAPTER – 6

REFERENCES

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REFERENCES:

Sandle, T. (2011). A Review of Cleanroom Microflora: Types, Trends, and Patterns. PDA journal of
Pharmaceutical science and Technology, 393-394.

sandle, T. (2014). Cleanroom Microbiology. United States: Davis Healthcare International Publishing.
USP. (2013). <1113> Microbial Characterization, Identification, and Strain Typing. United states
pharmacopeia, 784-785.

U.S. Pharmacopoeia-National Formulary [USP 9 NF 24]. Volume 30(5), United States

Pharmacopeial Convention. <1231> Water for Pharmaceutical use; p: 3056

2011 U.S. Pharmacopoeia-National Formulary [USP 29 NF 24], United States Pharmacopeial

Convention. <85> Bacterial Endotoxins Test; p: 2521

WHO. (2012). Test for bacterial Endotoxins.

Md. Sahab Uddin*, A. A. (2016). In-Process and Finished Products Quality Control, Tests for

Pharmaceutical Capsules According to. British Journal of Pharmaceutical Research, 1-2.

Mulay Sushruta*, K. A. (2011). An Overview of Limulus Amebocyte Lysate (LAL) test. International

Research Journal of Pharmacy, 67-68.

Sakhno NG, G. O. (2016). Microbial Identification Methods in Pharmaceutical Analysis: Comparison and

Evaluation. Mathews Journal of Pharmaceutical Science, 1

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