Pharmaegg.com, I am writing this Statement of Purpose to outline the purpose and procedures involved in the validation of endotoxin testing using the Gel Clot Method as per the requirements of the United States Pharmacopeia (USP) and other relevant regulatory guidelines. I am committed to ensuring the accuracy, reliability, and reproducibility of endotoxin testing in our laboratory setting. Through this SOP, I aim to establish clear guidelines and protocols for the validation of the Gel Clot Method, a widely accepted technique for bacterial endotoxin testing (BET).

PURPOSE

This SOP for BET Validation by Gel Clot Method has been established to provide the guidelines for the validation of Gel Clot Method in Microbiological lab to ensure the quality and purity of lysate.

This provides a test for estimating a concentration of LAL reagent that is present in the LAL reagent lysate. Limulus Amoebocyte Lysate (LAL) obtained from horse shoe crab (Limulus poly phemus).

The lysate extract from crab has ability to identify Bacterial Endotoxin. In Limit test Gel Clot Method when Endotoxin comes in contact with the lysate, it forms a firm gel.

The primary objective of this SOP is to validate the Gel Clot Method for endotoxin testing to guarantee the precision and consistency of results in compliance with regulatory standards. This SOP will ensure that our laboratory personnel are well-informed about the validation procedures and adhere to the established guidelines during routine endotoxin testing activities.

SCOPE

This procedure is applicable to microbiological section of Quality Control Lab. This SOP covers the validation procedures for the Gel Clot Method used in our laboratory for endotoxin testing of pharmaceutical products, medical devices, and related materials.

RESPONSIBILITIES & AUTHORITIES

Microbiologist is responsible for proper LAL validation testing by Gel Clot Method.

Quality Control Manager is responsible for implementation of this SOP.

REFERENCE & REQUIREMENTS

European Pharmacopeia

DEFINITIONS & ABBREVIATIONS

Endotoxin: are relatively heat stable, lipopolysaccharide protein complexes contained in cell walls of many fermentative, grams negative bacteria such as E. coli and klebsiella pneumonia.

Exotoxins: are heat labile protein complexes produced by certain gram-positive bacteria such as Staphylococcus aureus and Streptococcus Pyrogenes. Endotoxin and Exotoxins are liberated during cell death and lysis.

Gel Clot Technique: The lysate extract from crab has ability to identify Bacterial Endotoxin. In End Point Gel Clot Method when Endotoxin comes in contact with the lysate, it forms a firm gel.

PROCEDURE

Material And Equipment’s

1. Reaction Tubes
2. Incubator
3. Test Tube Rack
4. Pipettes
5. Non pyrogenic Test Tubes
6. Hot Air Oven with 250ºC
7. Non Pyrogenic plastic tips
8. Micro Pipettes
9. Control Standard Endotoxin (CSE)
10. LAL Reagent Water (LRW)
11. LAL multi test vial

Safety Measures

1. Avoid mouth pipetting
2. After completing work wash the hands and face with antiseptic soap.
3. Disposables should be tested for endotoxin contamination and interference (adsorption and/or extractable) with the LAL test.
4. Wear protective gloves and mask.
5. Don’t leave spatula with culture on the working bench without disinfecting it with IPA 70%.

Preparation Of Apparatus

All glass ware should be depyrogenated by exposing them at 250°C for 30 minutes in Hot Air Oven according to SOP.

Reconstitution Of Reagents

Confirmation of Lysate Sensitivity

Before using the lysate its sensitivity must be checked. To confirm the sensitivity of lysate (Pyrotell) the test must be carried on a series of known standard Endotoxin concentrations that bracket the labeled sensitivity (i.e. 2, 0.5λ and 0.25 λ)(λ denotes the actual claimed sensitivity of lysate) if λ = 0.25 EU /ml and Endotoxin is 2500/vial then Endotoxin concentrations should be made as follows:

Control Standard Endotoxin (CSE)

1. Control Standard Endotoxin (CSE) may be used to prepare controls for the Limulus Amoebocyte Lysate (LAL). The vials may appear to be empty, but on close examination, a fine web of endotoxin may be visible.
2. Reconstitute the CSE as follows:
3. Remove the metal seal from the vial and aseptically remove the stopper.
4. Add LRW to the vial. Recommended reconstitution volume is 5 ml, however, alternate volumes may be used to achieve desired concentration of stock solution.
5. To reconstitute with a pipette, break the vacuum by lifting the stopper just enough to allow air to enter, remove the stopper and add LRW.
6. Seal the vial with Parafilm
7. Vortex vigorously for one minute, at 5-10 minute intervals over a 30-60 minute period at room temperature.
8. Store reconstituted CSE at 2-8°C for not more than four weeks.
9. Do not freeze CSE.
10. Vortex the CSE for at least 30 seconds immediately before making the first dilution. And make dilution as follows:
11. Take 0.1ml from the stock CSE solution and add 0.9ml of LRW to obtain strength 50 EU/ml.
12. Take 0.1ml from 1st dilution and add 0.9ml LRW to get 5EU/ml.
13. Take 0.2ml from 2nd dilution and add 0.8ml LRW to obtain strength of 1.0EU/ml.
14. Take 0.5ml of 3rd dilution and add 0.5ml LRW to get strength of 0.5EU/ml.
15. Take 0.5ml from 4th dilution and add 0.5ml LRW to obtain 0.25EU/ml
16. Take 0.5ml from 5th dilution and add 0.5ml LRW to obtain 0.125EU/ml
17. Take 0.5ml from 6th dilution and add 0.5ml LRW to obtain 0.0625EU/ml
18. Vortex between dilutions.

Reagent (Lysate)

1. Gently tap the vial of LAL to cause loose LAL to fall to the bottom of the vial.
2. Remove the crimp seal and break the vacuum by lifting the gray stopper.
3. Do not contaminate the mouth of the vial.
4. Do not inject through or reuse the stopper.
5. A small amount of LAL left on the stopper will not affect the test.
6. Reconstitute the LAL with 5ml LAL Reagent Water.
7. The lyophilized LAL pellet will go into solution within few minutes.
8. Before use, gently mix the contents of the vial to ensure homogeneity. Mixing too vigorously may cause excessive foaming which can cause a loss of sensitivity.
9. Transfer 0.1ml of the reconstituted LAL into each depyrogenated glass test tubes placed in ice tray.
10. Cover them immediately with aluminum foil and store these test tubes at or below -20 °C.
11. Reconstituted LAL can be frozen once. Reconstituted LAL will retain activity for three months if frozen immediately after reconstitution and held at or below -20 °C.

Endotoxin Controls:

Positive Control: The positive control concentration should be 2λ, λ, 0.5λ and 0.25λ. Where λ is the concentration of LAL used.

Negative Controls: LRW negative controls should be included with each batch of specimens tested.

Test Performance

1. Add 0.1ml of each of positive control 2λ, λ, 0.5λ and 0.25λ into separate test tubes.
2. Already containing 0.1ml previously reconstituted and frozen LAL.
3. Place the reaction tubes in an incubator at 37 + 1 ºC for 60 + 2 minutes. The reaction begins when CSE is added into LAL but does not proceed at an optimum rate until the mixture reaches 37 ºC.
4. Do not disturb the tubes during the incubation period. The gel forming reaction is delicate and may be irreversibly terminated if the tubes are handled, agitated or vibrated.
5. Remove and read reaction tubes one at a time. Do not wipe the tubes dry or bump them against the side of the rack during removal. Invert the tube in one smooth motion at an angle of 180º; do not pause half way in the inversion unless it is obvious that the gel has not formed.
6. A positive test is indicated by the formation of a gel which does not collapse when the tube is inverted.
7. The validation of the test depends upon the lowest concentration of control standard endotoxin. The test is considered valid when lowest concentration shows a negative result.
8. The lowest concentration of the CSE that clots the lysate is the end point of the test.
9. Calculate the Geometric mean end-point concentration by using the following formula.
   antilog ∑e
   Geometric mean end-point concentration = ————-
   f
   ∑ e = the sum of the log endpoint concentrations of the dilution series used
   f = the number of replicate test tubes
10. The geometric mean end-point concentration is the measured sensitivity of the lysate solution (IU/mL). If this is not less than 0.5λ and not more than 2λ, the labelled sensitivity is confirmed and issued in the tests performed with this lysate.

REQUIRED DOCUMENTS:

1. Flow Diagram (Annexure A)
2. Sterility and LAL Test Log
3. LAL Test (Gel Clot Limit Method) Record.

Through the implementation of this SOP, our laboratory aims to establish the Gel Clot Method as a validated technique for endotoxin testing, ensuring the reliability and accuracy of results. This SOP will serve as a reference for all personnel involved in endotoxin testing activities, promoting consistency and adherence to regulatory standards.