Bacterial Endotoxin Test (BET or LAL Test) Method Validation

1.0 INTRODUCTION

Endotoxin from gram-negative bacteria are the most common cause of toxic reactions resulting from contamination of pharmaceutical products with pyrogens; their pyrogenic activity is much higher than that of most other pyrogenic substances. These bacterial endotoxins are lipopolysaccharides. Although there are a small number of pyrogens, which possess a different structure, the conclusion is generally justified that the absence of bacterial endotoxins in a product implies the absence of pyrogenic components, provided the presence of non-endotoxin pyrogenic substances can be ruled out.

The presence of endotoxins in a product may be masked by factors interfering with the reaction between the bacterial endotoxins and the amoebocyte lysate. Hence, the analyst who wishes to replace the rabbit pyrogen test required in a pharmacopoeial monograph by a test for bacterial endotoxins has to demonstrate that a valid test can be carried out on the product concerned; this may entail a procedure for removing interfering factors

2.0 OBJECTIVE

The Objective of this protocol is to establish documented evidence that the process employed for BET testing of Dextrose Injection IP (5% w/v) by Gel clot method will produce the desired results consistently when performed as per the standard operating procedures.

3.0 SCOPE

This protocol is applicable to Dextrose Injection IP (5% w/v), which requires endotoxin test.

4.0 RESPONSIBILITIES

S. No.	Responsibilities	Name of the Department
1	Development of Validation protocol	QC
2	Execution of this protocol	QC
3	Approval of protocol and of the final report	QA
4	Final determination of System Acceptability	AGM Works
5	Review and assembling of data into a final report	QC

5.0 PRE-REQUISITES

In order to efficiently conduct validation of the BET by Gel Clot method for the determination of Endotoxin content in Dextrose Injection IP (5% w/v), ensure that the following requirements are fulfilled.
1. Proper BET test Facility.
2. All instruments to be used for method validation are qualified and operational SOP’s established and followed.
3. Glassware should be cleaned, Micropipette should be accurate and dilution tubes should be properly depyrogenated.
4. Trained personnel for conducting BET test and its Validation.

6.0 EQUIPMENT / SYSTEM DESCRIPTION

1. Location: Quality Control department
2. Equipment: The equipment covers BET Heating Block, Hot air Oven.

7.0 MATERIALS AND METHODS

1. Depyrogenated glass tubes 10x75mm
2. Depyrogenated borosilicate glass tubes 16x125mm tubes.
3. Heating block set and maintained at 37ºC ± 1ºC prior to incubation.
4. Vortex Mixer.
5. Timer.
6. Sterile and depyrogenated fresh disposable pipette tips.
7. Calibrated Pipettes.
8. Test tube racks.
9. LAL reagent (Lysate).
10. Control Standard Endotoxin (CSE).
11. LAL reagent water.
12. Dextrose Injection IP (5% w/v)

8.0 PRODUCT DETAILS

All the bottles are sampled for BET Validation studies after terminal sterilization and the details are as under.

S. No.	Product Name	Batch No.	Batch Size	Date of sampling	Bottles sampled	Sampled by	Sign
1.
2.
3.

9.0 VALIDATION TEST

Validation of Bacterial Endotoxin test by gel clot method is done by following methods-
A. Test for Confirmation of Labeled LAL Reagent Sensitivity
B. Test for Interfering Factors

A. TEST FOR CONFIRMATION OF LABELLED LAL REAGENT SENSITIVITY 

PROCEDURE

1. Reconstitute Control Standard Endotoxin (CSE) by adding LRW according to manufacturer’s instruction. This reconstitute CSE will be the stock of CSE. Vortex the vial vigorously for 15 minutes.
2. Further dilute the stock solution to prepare a series of the standard solution of at least 4 concentrations equivalent to 2λ, λ, 0.5λ, and 0.25λ by diluting the standard endotoxin stock solution with LRW
3. Reconstitute the LAL reagent as per manufacturer's instruction.
4. Pipette 100 ml 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 blank use 100 ml LRW separately and perform the test in quadruplicate
5. Add 100 ml of reconstituted Lysate into each tube and mix gently.
6. Incubate all the tubes in block heater at 37 ± 1ºC for 60 ± 2 minutes. Start the timer immediately.
7. After 60 minutes incubation, read for gel formation by gently inverting the tubes at 180º with a single smooth action.
8. If a firm gel has formed that remains in place upon inversion, record the result as a positive. A result is negative if an intact gel is not formed
9. The endpoint is the last positive result in the series of decreasing concentration of Endotoxin. Calculate the mean value of the logarithms of the endpoint concentrations and then antilogarithm of the mean value using the following expression.
Geometric mean endpoint concentration = antilog Se/f
Se = Sum of the log endpoint concentration of the dilution series used,
f = number of replicates.

ACCEPTANCE CRITERIA

1. The test is not valid unless the lowest concentration of the standard solution shows a negative result in all replicate tests.
2. The geometric mean end-point concentration is the measured sensitivity of lysate solution (EU/ml or IU/ml). If this is not less than 0.5λ and not more than 2λ, the labeled sensitivity is confirmed.

B. TEST FOR INTERFERING FACTOR

PROCEDURE

1. Reconstitute Control Standard Endotoxin (CSE) by adding LRW according to manufacturer’s instruction. This reconstitute CSE will be the stock of CSE. Vortex the vial vigorously for 15 minutes.
2. Calculate MVD for sample specimen by following formula –

MVD = Endotoxin Limit X Potency of Product
                             Lysate Sensitivity

MVD = 0.5 EU/ml X 1ml/ ml
                 0.03EU/ml

MVD = 1: 16
MVD/4 = 1:4

3. Dilute the sample to MVD/4 i.e. 300µl sample and 900 µl LRW and shake on a vortex shaker for 3 minutes.
4. Prepare solution A, i.e. negative product control (NPC) by adding 50 ml Product sample at MVD/4, 50ml LRW and 100 ml of Lysate in a quadruplicate of 10 X 70 mm depyrogenated tube and label as a A1, A2, A3, and A4.
5. Prepare solution B, i.e. positive product control (PPC) by adding 50 ml of each Endotoxin standard of 4λ, 2λ, λ and 0.5λ to the depyrogenated assay tube which is labeled as B2λ, Bλ, B0.5λ and B0.25λ respectively in a quadruplicate. Add 50ml of the Product sample, which is diluted to MVD/4 in the labeled tubes of B2λ, Bλ, B0.5λ and B0.25λ respectively.
6. Prepare Solution C, i.e. Positive water control (PWC) by adding 50 ml of each Endotoxin standard of 4λ, 2λ, λ and 0.5λ to the depyrogenated assay tube, which is labeled as C2λ, Cλ, C0.5λ and C0.25λ respectively in a quadruplet. Add 50ml of LRW, to the labeled tubes of C2λ, Cλ, C0.5λ and C0.25λ respectively
7. Prepare solution D, i.e. negative water control (NWC) by adding 100 ml LRW and 100 ml of Lysate in a quadruplicate of 10 X 70 mm depyrogenated tube and label as a D1, D2, D3, and D4
8. Finally add 100 ml of Lysate to each tube starting with negative control and ending with highest Endotoxin concentration for solution A, solution B and Solution C

Table - I

Solution	Sample tube name	LRW	Product MVD/4	CSE	LAL	No of replicates
A (NPC)	-	50ml	50 ml	-	100µl	4
B (PPC)	2λ	-	50 ml	50 µl of 4λ	100µl	4
	λ	-	50 ml	50 µl of 2λ	100µl	4
	0.5λ	-	50 ml	50 µl of λ	100µl	4
	0.25λ	-	50 ml	50 µl of 0.5λ	100µl	4
C (PWC)	2λ	50 ml	-	50 µl of 4λ	100µl	4
	λ	50 ml	-	50 µl of 2λ	100µl	4
	0.5λ	50 ml	-	50 µl of λ	100µl	4
	0.25λ	50 ml	-	50 µl of 0.5λ	100µl	4
D (NWC)	-	100 ml	-	-	100µl	4

9. Prepare the same sample solution for all three batches as per table –I.
10. Incubate all the tubes in block heater at 37 ± 1ºC for 60 ± 2 minutes. Start the timer immediately.
11. After 60 minutes incubation, read for gel formation by gently inverting the tubes at 180º with a single smooth action.
12. If a firm gel has formed that remains in place upon inversion, record the result as a positive. A result is negative if an intact gel is not formed
13. Calculate the mean value of the logarithms of the endpoint concentrations for Solution B and Solution C and then antilogarithm of the mean value for both solution using the following expression:
Geometric mean endpoint concentration = antilog Se/f
Se = Sum of the log endpoint concentration of the dilution series used,
f = number of replicates.

ACCEPTANCE CRITERIA

1. The test is not valid unless all replicates of the solution A and D shows no reaction and the result of solution C confirms the labeled Lysate sensitivity.
2. The sensitivity of Lysate determined with solution B is not less than 0.5λ and not greater than 2λ, the test solution does not contain interfering factor under the experimental conditions used.

Precautions

1. Strict aseptic conditions must be maintained to avoid microbial contamination.
2. All containers and equipment must be Endotoxin-free by depyrogenating at 250ºC for 60 min. in a dry heat sterilizer.
3. Time and temperature regarding incubation should be maintained accurately.
4. No disturbance should be created during incubation.
Also see:
Bacterial Endotoxin Test Methods
Endotoxin Detection by Gel-Clot Methods
Endotoxin Detection by Quantitative Methods
Endotoxin Detection by Gel-Clot Limit Test Method
Endotoxin Detection by Semi-Quantitative Gel-Clot Method
Endotoxin Detection by Kinetic Thrbidimetric and Kinetic Chromogenic Methods
Endotoxin Detection by End-Point Chromogenic Method

Get ready to use editable documents in MS-Word FormatView List

Share

Tweet

Share

Share