1.0
Purpose:
1.1 To establish a formal procedure for the bacterial endotoxin
test procedure of Raw Material and Finished Product.
2.0
Scope:
2.1 This
SOP applies to Microbiological Laboratory.
3.0
Responsibilities:
3.1
Officer (Microbiology) is responsible for
performing the Bacterial Endotoxin test.
3.2 The Head of Quality Control/In-Charge is responsible for the compliance of this SOP.
3.3 Head of QA is responsible for SOP compliance.
4.0
Abbreviations
and Definitions
4.1 BET : Bacterial Endotoxin Test
4.2 CSE : Control Standard Endotoxin
4.3
MVD :
Maximum Valid Dilution
4.4
LRW :
Lal Reagent Water
4.5
PPC
:
Positive Product Control
4.6
NPC :
Negative Product Control
4.7
PWC :
Positive Water Control
4.8
NWC :
Negative water control
5.0
Materials
and Equipment
5.1 Equipment’s required:
5.1.1 LAFU
5.1.2 Incubator
5.1.3 Vortex mixture
5.1.4 Micropipettes
5.2 Material Required:
5.2.1 CSE
5.2.2 Lysate
5.2.3 LRW
5.2.4 Depyrogenated dilution tube
5.2.5 Depyrogenated assay tube
5.2.6 Sterile, endotoxin free micro tips
6.0 Precaution / Health and Safety Considerations
6.1 Ensure the depyrogenation of all glassware.
6.2 Ensure plastic apparatus are free of detectable Endotoxin or interfering effects of the test.
6.3 Always use fresh reconstituted lysate.
6.4 Rehydrated CSE may be stored for 28 days at 2 to 8°C.
6.5 Do not vortex lysate.
7.0
Procedure:
7.1
General:
7.1.1
Clean all the required glassware’s and depyrogenate at
220° C for 1.5 hours.
7.1.2
Transfer all the depyrogenated glassware’s to
microbiology lab.
7.1.3
Switch “ON” the Incubator and set the temperature at
37°C.
7.1.4
Calculate MVD of the product by following formula –
|
MVD = |
Endotoxin Limit X Product
Concentration |
|
Lysate
Sensitivity |
7.1.5
Prepare the sample according to its product
concentration.
7.1.6
Dilute the sample to be tested to its half MVD with
LRW and shake properly 3 minute on vortex mixer for.
7.2
Preparation Of Control Standard Endotoxin (CSE):
7.2.1 Reconstitute CSE in LRW as per manufacturer
instruction and vortex for 5 - 10 minutes.
7.2.2
Use Reconstituted CSE within 4 weeks after
reconstitutions. Store reconstituted CSE at 2°C to 8°C
temperature.
7.2.3
Prepare a CSE dilution with LRW to get 1 EU/ml and
vortex for 5 minutes.
7.2.4
Prepare a 4λ by diluting from 1 EU/ml with LRW and
vortex for 5 minutes (4λ to be back calculated from λ where λ is the Lysate
sensitivity).
7.3
Reconstitution of Lysate:
7.3.1
Reconstitute the lysate
after opening the aluminium seal of lysate.
7.3.2 Loosen lysate powder into
the bottom of the vial by tapping on a hard surface and then open the cork
slowly.
7.3.3 As per manufacturer’s
instruction, reconstitute with LRW before use and replace the cork immediately.
7.4
Confirmation of the Labelled Lysate Sensitivity:
7.4.1
Confirm in four replicates the labelled sensitivity λ, 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.
7.4.2
Prepare
CSE dilutions from 4λ
solutions of at least four concentrations equivalent to 2λ, λ,
0.5λ and 0. 25λ by diluting a series of CSE solution with water for
BET (LRW).
7.4.3 Take
10 depyrogenated assay tubes and label the tubes by numbering and arrange
quadruplicate in stand and the proceed as per following Table - 1
Table - 1
|
Sample
|
CSE
dilution
used |
LRW |
Lysate in µl |
No.
Of replicates |
|
2 λ |
100µl |
- |
100 µl |
4 |
|
λ |
100 µl |
- |
100 µl |
4 |
|
0.5
λ |
100 µl |
- |
100 µl |
4 |
|
0.25
λ |
100 µl |
- |
100 µl |
4 |
|
NWC |
- |
100 µl |
100 µl |
4 |
7.4.4 Pipette 100 µl diluted CSE i.e. to 2λ, λ, 0.5λ, and 0.25λ separately into depyrogenated borosilicate test tubes (10 mm X 75 mm) and labeled accordingly. For NWC (negative water control) use 100 µl LRW separately and perform the test in quadruplicate.
7.4.5
Add
100 µl of reconstituted Lysate into each tube and mix gently.
7.4.6
Incubate
the reaction mixture for a constant period at 37° c± 1° c for 60 ± 2 minutes,
avoiding vibration.
7.4.7 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.
7.4.8
The
test is not valid unless the lowest concentration of the standard solutions
shows a negative result in all replicate tests.
7.4.9
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:
7.4.10
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 0.5 λ and not more than 2 λ, the
labelled sensitivity is confirmed and is used in the tests performed with this
Lysate.
7.4.11
Test
Procedure:
7.4.12
Prepare
solutions A, B, C and D as shown in Table 2
Table – 2
|
Solution |
Solution description |
LRW in
µl |
42λ (CSE) in µl |
Product at MVD/2 in µl |
Lysate in µl |
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 |
7.4.13
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.
7.4.14
Add 50µl
of LRW in PPC and PWC, and 100 µl in NWC. Immediately add 50 µl of product
sample, which is diluted at MVD/2 in a NPC and PPC, and then add 50µl of CSE
that is diluted to 4λ in a PPC and PWC.
7.4.15 Finally add 100µl 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°C ± 1°C for 60 ± 2 minutes.
Flow
Sheet: This diagram illustrates the testing procedure of LAL test.
7.4.16 Reactive
Test: Take 100μl dilulated sample (as indicated) and then take 100μl LAL (0.125EU/ml) reagent into LAL test tube.
7.5
Interpretation of Result:
7.5.1
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°.
7.5.2
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°.
7.5.3
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.
7.5.4
The preparation being examined complies with the test
when a negative result is found for both replicates of solution A.
7.5.5
When
a positive result is found for both replicates of solution A, it does not
comply with the test.
7.5.6
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.
7.6
Failure Investigation:
7.6.1
When
positive result observed on both the tubes of test preparation, investigate the
cause of its failure by checking following parameters.
7.6.2
Check
product dilution, CSE dilution and lysate dilution.
7.6.3 Whether
Glassware’s are cleaned and dehydrogenated as per standard Operating procedure.
7.6.4
Check
sensitivity report of Lysate lot and matched CSE.
7.6.5
Check
Incubator temperature and calibration.
7.6.6
Check
Micropipette calibration.
7.6.7
Check
any source of contamination occur due to microbiologist.
8.0 Reference Document
8.1
USP <85>
9.0 Annexure
9.1
Annexure-I: Bacterial
Endotoxin Test Report
10.0
Revision
History
|
Revision
No. |
Brief reason for the revision |
Effective Date |
Remarks |
|
01 |
New |
|
|
11.0
Training
11.1
Head of Quality Control or his/her
nominee shall give the SOP training before effective date.
