SOP for Endotoxin Testing in Injectable Products (LAL Test)

Purpose

This SOP aims to outline the procedure for conducting endotoxin testing on injectable products to ensure compliance with regulatory requirements and patient safety.

Scope

This SOP applies to all injectable products manufactured within the facility that require endotoxin testing.

Responsibilities

QC, Microbiologist - To carry out the Bacterial Endotoxin Test in the Microbiology Laboratory

Production Department: Responsible for providing samples and supporting QC during testing.

Accountability

HOD - to ensure that the BET test is carried out once per month.

Definition

• Maximum valid dilution (MVD) - is the maximum allowable dilution of a specimen at which the endotoxin limit can be determined.
• Amoebocyte Lysate - lyophilized product obtained from the lysate of amoebocytes (white blood cells) from the horseshoe crab (Limulus polyphemus or Tachypleus.
• Endotoxin limit - the approximate threshold of pyrogens represented in; mg/ml in the case of endotoxin limit specified by weight (IU/mg) - units/ml in the case of endotoxin limit specified by unit of biological activity (IU/Unit) - ml/ml when the endotoxin limit is specified by volume (IU/ml)
• Lysate Sensitivity - measured optical density against known standard endotoxin concentration expressed in EU/ml or IU/ml.

Procedure

Material Requirements

• Amoebocyte Lysate
• Water for Bacterial Endotoxins Test (BET)
• Endotoxin standard
• Vortex mixer
• Glass pipettes depyrogenated
• Stopwatch
• Heating block
• Clean bench
• Test tubes (for assay) depyrogenated: 10 x 75mm
• Test tubes (for dilutions) depyrogenated: 20 x 150mm
• Alluminium foil depyrogenated
• Hot air oven
• Test tube stands
• Medical device: 10 pcs of each product
• Sterile Disposable Gloves
• 70% Isopropyl alcohol

Equipment Preparation

• Set up the endotoxin testing system according to the manufacturer’s instructions.
• Ensure all equipment, including the endotoxin-specific reagents, is properly calibrated and verified.
• Prepare the standard endotoxin solutions as required for validation.

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General

• Clean all the required glassware and depyrogenate in a hot-air oven at 250°C for 30 minutes.
• Transfer all the depyrogenated glassware to the Microbiology laboratory (BET room).
• The use of sterile gloves and related accessory testing is required.
• Switch on the heating block as per the SOP of operating instructions for the heating block and set the temperature at 37 ± 1°C.
• Calculate the MVD for the sample specimen by the following formula

MVD = ( endotoxin limit x concentration of Sample Solution/('"A)

• Dilute the sample to be tested to its one-fourth MVD with LWR and vortex for 3 minutes.

Lysate Sensitivity Confirmation

Confirm in four replicates the labeled sensitivity I, expressed in EU/ml or IU/ml, of the lysate solution before 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 that may affect the outcome of the test.

Sampling and Testing for Water for Injection

Collect the water for the injection sample in depyrogenated vials or test tubes as per the sampling plan. Testing water for injection is to be done per the method of analysis.

Preparation of the Standard Endotoxin Stock Solution

• The standard endotoxin stock solution is prepared from an endotoxin reference standard that has been calibrated against the International Standard, for example, endotoxin standard BRP. Endotoxin is expressed in International Units (IU).
  The equivalence in IU of the International Standard is stated by the World Health Organization.

NOTE: One International Unit (IU) of endotoxin is equal to one Endotoxin Unit (E.U.). Follow the specifications in the package leaflet and on the label for preparation and storage of the standard endotoxin stock solution.

Preparation of the Standard Endotoxin Solutions

• After vigorously mixing the standard endotoxin stock solution, prepare appropriate serial dilutions of this solution using water for bacterial endotoxins test (water for BET).
• Use the solutions as soon as possible to avoid loss of activity by adsorption.

Preparation of the Test Solutions

• Prepare the test solutions by rinsing medical devices using water for BET.
• If necessary, adjust the pH of the test solution ( or dilution thereof) so that the pH of the mixture of the lysate and test solution falls within the pH range specified by the lysate manufacturer.
• This usually applies to a product with a pH in the range of 6.0 to 8.0. The pH may be adjusted by the use of acid, base, or a suitable buffer, as recommended by the lysate manufacturer.
• Acids and bases may be prepared from concentrates or solids with water for BET in containers free of detectable endotoxin. Buffers must be validated to be free of detectable endotoxin and interfering factors.

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Test procedure

Prepare solutions A, B, C, and D as shown in table 1.

Solution	Solution Description	LRW (in µl)	4λ (CSE in µl)	Product at MVD/4 (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

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

Interpretation of the Results

• Each tube is interpreted as either negative or positive, the positive test indicates the formation of firm gel capable of maintaining its integrity when the test tube is inverted 180°C.
• A negative test is characterized by the absence of gel or by the formation of viscous mass, which does not hold when the tube is inverted at 180°C
• The test is not valid unless both replicates of the two positive control solutions Band Care positive and those of the negative control solution D are negative.
• The preparation being examined complies with the test when a negative result is found for both replicates of solution A and it does not comply when a positive result is found for both replicates.
• Repeat the test if a positive result is found for one replicate of solution A and a negative result is found in the other.

Acceptance Criteria

• Endotoxin levels in injectable products should not exceed the specified limits for safe administration.
• Ensure that all controls demonstrate appropriate responses within established limits.

Documentation

• Record all endotoxin testing results, including method validation data, in the Endotoxin Testing Record.
• Maintain accurate records of sample preparation, testing conditions, and any deviations encountered.
• Review and approve the documentation by the QC Manager.

Abbreviations

SOP: standard operating procedure

QA: Quality Assurance

QC: Quality Control

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

Annexure

Nil

Revision History

Nil

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