Bacterial Endotoxin Test

Dr. Syeda Rima Ishaq

USP Consultant
6 July, 2021
Regulatory Requirement

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Regulatory Requirement

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Regulatory Requirement

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Endotoxin
• What is it ?
• Where does it come from ?
• Why is it important?

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Bacterial Endotoxin Test Application

• Injectable drugs
• medical devices which are in
contact blood or spinal fluid
• Raw materials,
• Water and
• in process monitoring

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Bacterial Endotoxin Test Application
• Cyanocobalamin Inj., can have an endotoxin concentration of
10 EU/ml and be considered as in compliance.
• Water for Injection (WFI) System should have a level of
endotoxin NMT 0.25 EU/ml.

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Endotoxin testing
• Pyrogen Test on Rabbit
• Bacterial Endotoxin Test

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Bacterial Endotoxin Test
• Developed in 1960s by Drs. Bang and Levin
• Based on clotting reaction of horseshoe crab
blood to endotoxin
• Faster, more economical, more sensitive than
rabbit pyrogen test

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Bacterial Endotoxin Test
• Limulus - genera of crab
• Amebocyte - crab blood cell from which
active component is derived
• Lysate - component is obtained by separating
amebocytes from the plasma and then lysing them

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Principle of the test

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Types of Methods
• There are four basic methods commercially available and
currently approved by FDA for end-product release testing:
• (i) the gel-clot;
• (ii) the turbidimetric (spectrophotometric);
• (iii) the colorimetric (Lowry protein); and
• (iv) the chromogenic

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Gel Clot Method
• The gel-clot technique is for
detecting or quantifying
endotoxins based on clotting of
the lysate TS in the presence
of endotoxin.
• Lowest reagent Cost
• Simple Reporting
• Found in all pharmacopoeia
• Good stability
• Simple technique

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Disadvantages
• Limit test
• Least sensitive
• Time consuming
• Poor trending options
• Variability

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False results
• Use of wrong concentration
• Use of improper dilution and dilution tubes
• Wrong incubating temperature
• Improper storage of Lysate and CSE
• Contaminated diluent

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Glass wares
• Depyrogenate all glassware and other heat stable materials in a
hot air oven using a validated process.
• If employing plastic apparatus such as microplates and pipet
tips for automatic pipetters, use apparatus shown to be free of
detectable endotoxin and which does not interfere in the test.

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Preparation of Standard Endotoxin
Stock Solution
• A Standard Endotoxin Stock Solution is prepared from an endotoxin
reference standard that has been calibrated against the International
Standard for endotoxins.
• One International Unit (IU) of endotoxin is equal to one Endotoxin
Unit (EU).
• Add water for injection as per manufacturer instrusction.
• Mix vigorously.
• prepare appropriate serial dilutions of Standard Endotoxin Solution,
using water BET.
• Use dilutions as soon as possible to avoid loss of activity by
adsorption.

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Test for confirmation of labeled lysate
sensitivity
• Confirm in four replicates the labeled sensitivity, λ, expressed in
IU/ml of the lysate prior to use in the test.
• The test for confirmation of the lysate sensitivity is to be carried
out when a new lot of lysate is used or when there is any
change in the test conditions which may affect the outcome of
the test.

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Test for confirmation of labeled lysate
sensitivity
• Prepare standard solutions having at least four concentrations
equivalent to 2λ , λ, 0.5λ and 0.25λ .
• Mix a volume of the lysate TS with an equal volume of one of
the standard solutions (such as 0.1 ml aliquots) in each tube
• Incubate the reaction mixture for a constant period according to
directions of the lysate manufacturer (usually at 37±1°C for 60 ±
2 minutes), avoiding vibration.

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Test for confirmation of labeled lysate
sensitivity
• Test the integrity of the gel for tests carried out in tubes, take
each tube in turn directly from the incubator and invert it through
approximately 180 degrees in one smooth motion.
• If a firm gel has formed that remains in place upon inversion,
record the result as positive. A result is negative if an intact gel
is not formed.

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Preparation of sample solutions
• Prepare sample solutions by dissolving or diluting drugs using
water BET.
• If necessary, adjust the pH of the solution to be examined (or
dilution thereof) so that the pH of the mixture of the lysate TS
and sample solution falls within the pH range specified by the
lysate manufacturer, usually 6.0 to 8.0.
• Acids and bases may be prepared from concentrates or solids
with water BET in containers free of detectable endotoxin.
• Buffers must be validated to be free of detectable endotoxin and
interfering factors.

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DETERMINATION OF MAXIMUM VALID
DILUTION
• The Maximum Valid Dilution (MVD) is the maximum allowable
dilution of a sample at which the endotoxin limit can be
determined. Determine the MVD from the following equation:
• MVD = Endotoxin Limit × Concentration of sample solution /λ

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Endotoxin Limit
• The effects of endotoxin are related to the amount of endotoxin
in the product dose administered to a patient.
• the endotoxin limit is expressed as K/M. K is 5.0 EU/kilogram
(kg.), which represents the approximate threshold pyrogen dose
for humans and rabbits.
• M represents the rabbit pyrogen test dose or the maximum
human dose per kilogram that would be administered in a single
one hour period, whichever is larger.

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Example 1
• A non-intrathecal drug product that has a maximum human
dose of 10 ml/kg.
• Endotoxin limit
• 0.5 EU/ml / 10 ml/kg = K 5 EU/kg

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Example 2
• Product: Cyanocobalamin Inj. Potency: 1000 mcg/ml
• Maximum Dose/kg 14.3 mcg/kg
• Endotoxin Tolerance Limit = 5.0 EU/kg - non-intrathecal drug
• Endotoxin Limit = K 5.0 EU/kg/M 14.3 mcg/kg =0.35 EU/mcg
0.35 EU/mcg) is expressed in Endotoxin Units per mcg of
product. In order to convert this value to a endotoxin unit
concentration per ml, multiply it (0.35 EU/mcg) by the product
potency (see below).
• 1000 mcg/ml x 0.35 EU/mcg = 350 EU/ml

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Validation
• Inhibition
• Enhancement
• Non Interference

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Inhibition and Enhancement
• Most of the inhibition can be overcome by dilution of the
product.
• Other factors such as the shape and type of glassware used in
the gel-clot test can also affect the validity of the test.
• For example, siliconized glassware as well as plastic can inhibit
gel-clot formation or prevent accurate spectrophotometric
readings of the reaction mixture end point.
• Turbidimetric and chromogenic methods cannot be used with
certain turbid or colored products.

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Inhibition and Enhancement
• Additionally, precipitate formation, although inhibitory, may be
mistaken for a positive response in these methods.
• One problem associated with the use of the chromogenic
method is the formation of a precipitate following the addition of
acid to stop color development.
• Products that require a neutral or basic pH for solubility are
most likely to cause this problem.

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Inhibition and Enhancement
• several researchers have found that even the selection of lysate
reagent source (i.e., the manufacturer of the lysate) can
contribute to variability in test results. Therefore, if any change
in reagent source is made, the test must be re-validated.

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Test for interfering factors
• Usually prepare the solutions (A-D) in Table 1, and perform the
inhibition/enhancement test on the sample solutions at a dilution
less than the MVD not containing any detectable endotoxins,
operating as described above under Test for confirmation of
labeled lysate sensitivity

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