BET: Bacterial Endotoxin (LAL) Test : Pharmaceutical Guidelines

BET: Bacterial Endotoxin (LAL) Test

Learn how to conduct the Bacterial Endotoxin (LAL) Test for Injectables and Water fir Injections (WFI).

1.0  Equipment Required

       LAF
       Heating Block
       Cyclo mixture
       Micropipettes

2.0  Material Required

       CSE
       Lysate
       LRW
       Depyrogenated dilution tube 12mm X 75mm
       Depyrogenated assay tube 10mm X 75mm.
       Individually packed Sterile, endotoxin free micro tips

3.0  Procedure:

3.1  General

3.1.1  Clean all the required glassware and depyrogenate as per SOP for depyrogenation of glassware for BET.
3.1.1  Transfer all the pyrogen free glassware to BET Room.
3.1.2  Switch “ON” the heating block as per SOP for operating Instruction for Heating Block and set the temperature at 37°C.
3.1.3  Calculate MVD for sample specimen by following formula –

              Endotoxin Limit X Potency of Product
MVD = ---------------------------------------------
                       ʎ, Lysate Sensitivity

3.1.4  Dilute the sample to be tested to its half MVD with LRW and shake on vortex shaker for 3 minutes.

3.2  Preparation Of Control Standard (CSE):

3.2.1  Reconstitute CSE in LRW as per manufacturer's instruction as laid down in the bottle and certificate supplied by manufacturer, and vortex for 5 - 10 minutes
3.2.2  Use Reconstituted CSE within 4 weeks after reconstitutions. Store reconstituted CSE at 2°C to 8°C temperature.
3.2.3  Prepare a CSE dilution with LRW to yield 1EU/ml and shake the dilution on vortex for 5 minutes.
3.2.4  Prepare a 4ʎ by diluting a volume, which is equivalent to 4ʎ concentration from 1 EU/ml to LRW. Shake the dilution on vortex for 5 minutes. (4ʎ to be back calculated from lʎ is the Lysate sensitivity)

3.3  Reconstitution of Lysate:

3.3.1  Now reconstitute the lysate by opening the aluminium seal of lysate with the help of sterile blade.
3.3.2  Collect lysate powder into the bottom of the vial by tapping on a hard surface. And then open the cork slowly.
3.3.3  As per manufacturer's instruction, add LRW before use and replace the cork immediately.

3.4  Confirmation of The Labelled Lysate Sensitivity

3.4.1  Confirm in four replicates the labelled sensitivity l, expressed in EU/ml or IU/ml, of the lysate solution prior to use in the test. Confirmation of the lysate sensitivity must be carried out when a new batch of lysate is used or when there is any change in the experimental conditions which may affect the outcome of the test
3.4.2  Prepare CSE dilutions from 4ʎ solutions of at least four concentrations equivalent to 2ʎʎ  0.5ʎ and 0.25ʎ by diluting a series of CSE solution with water for BET (LRW)
3.4.3  Take 10 depyrogenated assay tubes and label the tubes by numbering and arrange quadruplicate in stand and the proceed as per following table  -
Sample
CSE dilution used
LRW
Lysate in ml
No. of replicates
2ʎ
100ml of 2ʎ
-
100 ml
4
ʎ
100 ml of ʎ
-
100 ml
4
0.5ʎ
100 ml of 0.5ʎ
-
100 ml
4
0.25ʎ
100 m of 0.25ʎ
-
100 ml
4
NWC
-
100 ml
100 ml
4
3.4.4  Pipette 100 ml diluted CSE i.e. to 2ʎʎ  0.5ʎ, and 0.25ʎ separately into depyrogenated borosilicate test tubes (10 mm X 75 mm) and labeled accordingly. For NWC (negative water control) use 100 ml LRW separately and perform the test in quadruplicate
3.4.5  Add 100 ml of reconstituted Lysate into each tube and mix gently
3.4.6  Incubate the reaction mixture for a constant period at 37° ± 1° for 60 ± 2 minutes, avoiding vibration.
3.4.7  After incubation, take each tube and turn directly from the incubator and invert it through approximately 180° 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
3.4.8  The test is not valid unless the lowest concentration of the standard solutions shows a negative result in all replicate tests
3.4.9  The endpoint is the last positive result in the series of decreasing concentrations of endotoxin. Calculate the mean value of the logarithms of the end-point concentrations and then the antilogarithm of the mean value using the following expression:
Lysate Sensitivity
3.4.10 The geometric mean end-point concentration is the measured sensitivity of the lysate solution (EU/ml or IU/ml). If this is not less than 0.5ʎ and not more than 2ʎ, the labelled sensitivity is confirmed and is used in the tests performed with this Lysate.

3.5  Test Procedure:

3.5.1  Prepare solutions A, B, C and D as shown in Table 1,

Table – 1

Solution
Solution description
LRW in ml
4ʎ (CSE) in ml
Product at MVD/2 in ml
Lysate in ml
No. of Replicates
A
Negative product control (NPC)
50
-
50
100
2
B
Positive Product control (PPC)
-
50
50
100
2
C
Positive water control (PWC)
50
50
-
100
2
D
Negative water control (NWC)
100
-
-
100
2

3.5.2 Take 8 depyrogenated assay tubes and label the tubes by numbering and arrange in stand one opposite to each other. i.e. 1 & 2 for NPC, 3 & 4 for PPC, 5 & 6 for PWC, 7 & 8 for Negative water control.
3.5.3 Add 50ml of LRW in PPC and PWC, and 100 ml in NWC. Immediately add 50 ml of product sample, which is diluted at MVD/2 in a NPC and PPC, and then add 50ml of CSE that is diluted to 4ʎ in a PPC and PWC.
3.5.4 Finally add 100ml of Lysate in all tubes and next, mix the assay tubes by hand and incubate in heating block, where the temperature is maintained at 37 ± 1°C for 60 ± 2 minutes.

3.6 Interpretation of Result:

3.6.1 Each tube in the gel clot method is interpreted as either positive or negative, Positive test indicates the formation of firm gel capable of maintaining its integrity when the test tube is inverted 180°.
3.6.2 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 at 180°.
3.6.3 The test is not valid unless both replicates of the two positive control solutions B and C are positive and those of the negative control solution D are negative.
3.6.4 The preparation being examined complies with the test when a negative result is found for both replicates of solution A.
3.6.5 When a positive result is found for both replicates of solution A, it does not comply with the test
3.6.6 Repeat the test if a positive result is found for one replicate of solution A and a negative result is found for the other. The preparation being examined complies with the test if a negative result is found for both replicates of solution A in the repeat test.

3.7 Failure Investigation:

3.7.1 When positive result observed on both the tubes of test preparation, investigate the cause of its failure by checking following parameters.
3.7.2 Check product dilution, CSE dilution and lysate dilution.
3.7.3 Whether glassware are cleaned and dehydrogenated as per SOP.
3.7.4 Check sensitivity report of Lysate lot and matched CSE.
3.7.5 Check Heating block temperature and calibration.
3.7.6 Check micropipette calibration.
3.7.7 Check any source of contamination occur due to microbiologist.

4.0 Precaution

4.1 Always use fresh reconstituted lysate.
4.2 Rehydrated CSE may be stored for 28 days at 2 to 8°C.
4.3 The supplied COA is specific for lysate lot and CSE lot. So use the same lot for testing.
4.4 Do not vortex lysate.

5.0 Frequencies

5.1 Batch wise.
5.2 Daily for WFI from all user points.
5.3 Confirmation of Lysate sensitivity – Each lot of consignment.

6.0 Abbreviation:

SOP : Standard Operating Procedure
BET : Bacterial Endotoxin Test
CSE : Control Standard Endotoxin
MVD : Maximum Valid Dilution
LRW : Lal Reagent Water
PPC : Positive Product Control
NPC : Negative Product Control
PWC : Positive Water Control
NWC : Negative water control





Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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1 comment: Post Yours! Read Comment Policy ▼

  1. Why we use double cse concentration (0.5EU) in PPC and PWC but lysate sensitivity is 0.125EU

    ReplyDelete

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