in parenteral products and water for injection (WFI).

2. Scope

This SOP applies to the Analytical Method Development (AMD) team for the development and optimization of BET methods applicable to injectable formulations, bulk solutions, and pharmaceutical-grade water systems.

3. Responsibilities

• Microbiologist: Performs the BET, documents the method development and interference testing.
• Analytical Scientist: Coordinates sample preparation and dilution strategies during development.
• QA Officer: Ensures all development and validation data meet regulatory and GMP expectations.
• Head – AMD: Reviews and approves final test protocol and development report.

4. Accountability

The Head of Analytical Method Development is accountable for method integrity, compliance with pharmacopeial requirements, and scientific accuracy of the BET development process.

5. Procedure

5.1 Reference Guidelines and Sensitivity Selection

1. Refer to:
   • USP <85> – Bacterial Endotoxins Test
   • EP 2.6.14 / IP BET chapter
2. Select an appropriate sensitivity (λ) based on endotoxin limit calculation:
3. Endotoxin Limit (EU/mL) = K / M
   • K = Threshold pyrogenic dose of endotoxin per kg body weight (e.g., 5 EU/kg)
   • M = Maximum dose per kg body weight
4. Document calculation in Annexure-1: Endotoxin Limit Determination Sheet.

5.2 Reagent Preparation and Controls

1. Reconstitute LAL reagent and control standard endotoxin (CSE) using LAL Reagent Water (LRW).
2. Prepare dilution series of CSE to bracket the selected λ.
3. Include:
   • Positive product control (PPC)
   • Negative control (LRW)
   • Inhibition/enhancement controls
4. Record all reagent and dilution preparations in Annexure-2: BET Reagent Preparation Log.

5.3 Method Selection and Validation

1. Choose the suitable technique:
   • Gel-Clot Method: For routine qualitative testing
   • Turbidimetric Method: For quantitative and high-throughput samples
   • Chromogenic Method: For low endotoxin detection and kinetic evaluation
2. Perform initial qualification of the method using sample dilution, recovery, and limit of detection.
3. Log validation results in Annexure-3: Method Suitability and Recovery Log.

5.4 Interference Testing

1. Mix test sample with CSE at various dilutions (e.g., 1:2, 1:4, 1:8) to assess interference.
2. Confirm that recovery falls within 50–200% of theoretical value.
3. If recovery fails, use additional dilution, depyrogenated glassware, or modify sample pretreatment.
4. Document in Annexure-4: Interference Test Summary.

5.5 BET Execution (Gel-Clot Method)

1. Dispense 0.1 mL of test sample or control into each LAL reagent tube.
2. Incubate at 37±1°C for 60±2 minutes undisturbed.
3. After incubation, invert tubes 180°.
4. A firm gel indicates positive; no gel or partial gel is negative.
5. Interpret result against standard endotoxin concentration series.
6. Record in Annexure-5: BET Raw Data Sheet.

5.6 Reporting and Finalization

1. Compile method development data, recovery results, interference assessment, and controls.
2. Summarize results and determine method suitability.
3. Prepare final development protocol for QA approval and implementation.
4. Document in Annexure-6: BET Method Development Report.

6. Abbreviations

• BET: Bacterial Endotoxins Test
• LAL: Limulus Amebocyte Lysate
• CSE: Control Standard Endotoxin
• LRW: LAL Reagent Water
• SOP: Standard Operating Procedure

7. Documents

1. Endotoxin Limit Determination Sheet – Annexure-1
2. BET Reagent Preparation Log – Annexure-2
3. Method Suitability and Recovery Log – Annexure-3
4. Interference Test Summary – Annexure-4
5. BET Raw Data Sheet – Annexure-5
6. BET Method Development Report – Annexure-6

8. References

• USP <85> – Bacterial Endotoxins Test
• EP 2.6.14 – Bacterial Endotoxins
• ICH Q6A – Specifications

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Endotoxin Limit Determination Sheet

Product	K Value	Maximum Dose	Limit (EU/mL)
Injection X	5 EU/kg	10 mL/kg	0.5

Annexure-2: BET Reagent Preparation Log

Reagent	Lot No.	Reconstitution Volume	Prepared By
LAL Reagent	LAL-0525	2.6 mL	Sunita Reddy

Annexure-3: Method Suitability and Recovery Log

Sample	Spiked Level	Recovery (%)	Status
Injection X	0.5 EU/mL	105%	Pass

Annexure-4: Interference Test Summary

The recovery across all dilutions ranged between 91–112%. No inhibition or enhancement observed. Sample matrix compatible with BET method.

Annexure-5: BET Raw Data Sheet

Tube ID	Sample	Gel Formation	Interpretation
GL-01	Test Sample	Yes	Positive
GL-02	LRW (Control)	No	Negative

Annexure-6: BET Method Development Report

The gel-clot method was found suitable for Injection X at an endotoxin limit of 0.5 EU/mL. All controls passed. Interference and recovery criteria met. Method suitable for validation.

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Incorporated chromogenic and turbidimetric options; added interference criteria	Annual Review