sop for BET (Bacterial Endotoxin) test in Microbiology

1.0 OBJECTIVE

1.1 To down the bacterial endotoxin in WFI & finished pharmaceutical product.

2.0 SCOPE

2.1 This procedure applicable for Bacterial Endotoxin Test in Microbiology Laboratory .

3.0 RESPONSIBILITY

3.1 Microbiologist QC

4.0 ACCOUNTABILITY

4.1 QC Manager

5.0 PROCEDURE

5.1 MATERIALS REQUIRED:
5.1.1 LAL Reagent Water, Control Standard Endotoxin, LAL Reagent, Depyrogenated Micro tips (100 μl &1000 μl), Glass test tube (depyrogenated), Sample to be tested.

5.2 EQUIPMENTS REQUIRED:
5.2.1 Micro pipettes (1000 μl &100 μl ), Vortex Mixer, Heating block
5.3 PROCEDURE FOR TESTING:
5.4 Preparation of Maximum valid dilution:
5.4.1 When the endotoxin limit in the substance or preparation being examined is specified in terms of
weight or units of active drug, the MVD may be calculated by the following formula:

Endotoxin Limit (in EU/ ml /mg) X Potency of product

MVD = ———————————————————————

Labeled sensitivity of lysate in EU /ml (l)

Where Potency =Concentration of the drug in mg / ml or unit /ml
5.4.2 When the endotoxin limit in the sample preparation is specified in terms of volume, the MVD may be
calculated by the following formula.

Endotoxin Limit (EU/ml)
MVD = —————————————————–
Labeled sensitivity of lysate in EU/ml (λ)

5.4.3 Dilute the sample MVD/2 times with LAL water when performing the test at MVD. If performing the test
at MVD/2, dilute the sample MVD/4time with LAL water.

5.5 Preparation of E-Coli Control Standard Endotoxin (CSE):
5.5.1 Reconstitute the CSE with appropriate volume (as stated on certificate) of LAL reagent water vortex
continuous 30 minutes at the interval of 10minutes (When new bottles reconstitute) & vortex vigorously
for five minutes before further dilution
5.6 Preparation of CSE dilution series:
5.6.1 Confirm the labeled sensitivity using at least one vial of each batch/lot of lysate. Prepare a series
of two-fold dilution of the CSE to give concentrations of 2λ, λ, 0.5λ and 0.25λ, where λ is the labeled
sensitivity of the lysate in EU per ml.
5.6.2 Perform the test as given as under Procedure on these four standard concentrations in duplicate or
greater in include negative control consisting of water BET.
5.6.3 Calculate the average of the logarithms of the lowest concentration of endotoxin in each series of
dilutions for which a positive result is found. The antilogarithm of this average gives the estimated
lysate sensitivity, which must be greater than or equal to 0.5λ and less than or equal to 2.0λ.
Confirm the labeled sensitivity of each new batch of lysate prior to use in the test. Test CSE results
are only valid when the positive water and specimen controls are positive at the 2 λ & λ endotoxin concentration.

5.7 Preparation of LAL Reagents:
5.7.1 Test LAL reagent for its sensitivity at the time of receiving. For reconstitution, Collect LAL powder into the bottom of the vial by tapping on a firm surface, unseal and release the vacuum by slowly lifting the
stopper, avoiding touch contamination. The small amount of LAL on the stopper is insignificant.
5.7.2 Rehydrate the reagent with appropriate volume of (as stated on label and certificate) LAL reagent water
by pipetting directly into the vial immediately before use. Cover the vial with an endotoxin free surface or
the inner side of perafilm when not in immediate use. Gently swirl until LAL dissolves into a colorless
solution.

5.8 Test procedure for gel clot:
5.8.1 Each assay should include dilutions of a sample or product, a negative water control, a positive
product control, and either a 2λ water control or a series of two-fold dilutions from an endotoxin
standard which bracket the labeled LAL sensitivity.

5.9 Interpretation of results:
5.9.1 Each tube in the gel-clot method is interpreted as either positive or negative. A positive test is defined as
formation of a firm gel capable of maintaining its integrity when the test tube is inverted 180°C. A negative
test is characterized by the absence of gel or by the formation of a viscous mass, which does not hold
when the tube is inverted. Calculate sample endotoxin concentration by the following formula.

Sample Endotoxin Conc. = Dilution Factor x Sensitivity of λ/ Conc. of Sample (Potency)
5.10 Re-tests:
5.10.1If the positive result is found for one of the test duplicates and a negative result for the other, the test
should be repeated as described above. The results of the retest should be interpreted as for the initial
test.
5.10.2The substance or preparation being examined meets the requirements of the test if the concentration of
endotoxin is less than the endotoxin limit stated in the monograph.
5.11 PRECAUTIONS:
5.11.1Vortex standard Endotoxin for not less than 30 minutes by taping vial to vortex mixture.
5.11.2Vortex all Endotoxin dilution for not less than 1 minute.
5.11.3Do not allow dilution of Endotoxin to stand for more than 10 minutes without re-vortexing
5.11.4Reconstitute Lysate very gently and mix carefully so as to avoid the formation of bubbles.
5.11.5Incubate tube in heating block kept on a stable surface, which do not get vibration, shake or bump.
5.11.6Do not transfer or disturb the tube at any stage of incubation till reading results.
5.11.7Observe result by inverting the tube very gently with constant speed.
5.11.8Observe result only once. Gel is fragile & may break when handled.
5.11.9Start the heating block before around 30 minutes before starting the test to get the temperature stabilize
at the time of incubation.

6.0 ABBREVIATIONS

Abbreviation used Extended Form
SOP : Standard Operating Procedure
QA : Quality Assurance
MB : Microbiology Lab
QC : Quality Control
NPC : Negative Product Control
PPC : Product Positive Control
PC : Positive Control
λ : Sensitivity Of LAL Reagent
EU : Endotoxin unit
MVD : Maximum Valid Dilution
LRW : LAL Reagent Water
LAL : Limulus Amoebocyte Lysate
CSE : Control Standard Endotoxin

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