SOP for bacterial endotoxin test is described in this post which you can follow in the section of microbiological lab.

Objective:

This procedure has been established for providing the guidelines for bacterial endotoxin test (BET) for aseptically manufactured products.

Scope:   

This procedure is applicable to microbiology lab.

Responsibility:                          

Microbiologist.                             

Accountability:

Quality Control Manager.            

Procedure:

For performing bacterial endotoxin test (BET) always use cleaned depyrogenated glasswares. 

Reconstitution of Control Standard (CSE)


Dilution

Sample

LRW

Final Concentration

First of all reconstitute the vial with 10ml of LRW to yield 1000eu/ml

1.    

1ml from solution of CSE+ 9ml= 1000EU/10ml

100EU/ml

2.    

1ml from DIL 1 + 9ml LRW=100EU/10ml

10EU/ml

3.    

1ml from DIL 2 + 9ml LRW=10EU/10ml

1EU/ml

4.    

1ml from DIL 3 + 2ml LRW=1EU/2ml

0.5EU/ml

5.    

1ml from DIL 4 + 2ml LRW=0.5EU/2ml

0.25EU/ml

6.    

1ml from DIL 5 + 2ml LRW=0.25EU/2ml

0.125EU/ml

First of all read the certificate of analysis properly provided with CSE vial and then reconstitute the vial with lal reagent water (LRW) as written in manufacturer/supplier specifications.

For example if single vial of CSE contains 1000 endotoxin units then dilute it with 10ml of LRW yielding the concentration as 100eu/ml in first dilution followed  serial dilution to yield 0.25eu/ml or 0.125eu/ml or your required lysate sensitivity. 

Vortex each dilution atleast for 5-10 minutes and use lysate  within 4 weeks after reconstitutions and store this solution at 2oC-8oC for 28days.

Confirmation of the Labeled Lysate Sensitivity: 

Always confirm the lysate sensitivity  prior to use of each new lot.

LRW (lal reagent water) will e used as negative control. 

Take 2 different lysate ampoules that are to be confirmed for their sensitivity  and add about 0.2ml of water for injection in it mix gently and then place in for incubation along with negative control (LRW) and positive control (CSE with your required sensitivity) samples separately for a constant period at 37° ± 1° for 60 ± 1 mint, avoiding vibration. 

At the end of incubation pick each ampoule and invert it at an angle of 180° in one smooth motion if on moving the solution does not move and shows a clot or pure gel formation then the result is positive and if the solution moves upon inverting the ampoule then the result is negative. 

 Sample Preparation:

Calculate the maximum valid dilution and reconstitute the sample if it is in dry powder form. 

Reconstituted Lysate should be clear. Do not use the Lysate if it contains gross particulate or color changes to yellow.

Allow the Lysate to equilibrate to room temperature prior to use.

Calculation of Maximum Valid Dilution (MVD)


 MVD= EL × concentration of product

                                  Λ

Whereas:


EL = Endotoxin limit as specified in the individual product monograph




Potency of product = Concentration of product in units or mg/ml







λ= Labelled sensitivity lysate


Now use the diluted sample for adding in lysate ampoule then swirl the ampoule gently and set for incubation at 37oC for 60±1minute.

At the end of incubation pick each ampoule and invert it at an angle of 180° in one smooth motion if on moving the solution does not move and shows affirm gel clot formation then the result is positive and if the solution moves upon inverting the ampoule then the result is negative. 

Result Interpretation:

There should be no gel clot present in the sample

There should be no gel clot present in the negative control.

 Gel clot should be present in the positive control.