to injectable products, water for injection (WFI), bulk drug substances, and other sterile/non-sterile samples analyzed for endotoxin content in the Analytical Method Development (AMD) laboratory.

3. Responsibilities

• Microbiologist/Analyst: Prepares reagents, standards, and samples and performs the kinetic-turbidimetric assay.
• Instrument Operator: Operates the endotoxin detection system and maintains calibration and control logs.
• QA Executive: Reviews data and ensures compliance with pharmacopeial limits and instrument suitability.

4. Accountability

The Head of Analytical Method Development is accountable for ensuring validated performance of endotoxin limit tests using appropriate LAL methods in accordance with USP, Ph. Eur., and IP guidelines.

5. Procedure

5.1 Reagents and Standards

1. Use LAL reagent suitable for kinetic-turbidimetric method (e.g., PyroGene™, Endosafe™).
2. Prepare Control Standard Endotoxin (CSE) as per Certificate of Analysis instructions.
3. Use endotoxin-free glassware, pipette tips, and water (LAL Reagent Water).

5.2 Instrument Preparation

1. Switch on the endotoxin detection system and allow stabilization (e.g., BioWhittaker Kinetic-QCL Reader).
2. Run self-check and ensure calibration and software validation are within due dates.

5.3 Sample Preparation

1. Reconstitute or dilute samples in endotoxin-free conditions using LAL water.
2. Perform initial inhibition/enhancement testing to determine valid dilution (Annexure-1).
3. Filter or centrifuge samples if turbidity or insoluble matter is present.

5.4 Assay Procedure

1. Prepare standard curve using at least three concentrations of CSE (e.g., 0.5, 1.0, 2.0 EU/mL).
2. Inoculate microplate wells with 100 µL each of sample/standard + 100 µL of LAL reagent.
3. Run the kinetic-turbidimetric assay for 60–90 minutes with continuous optical density reading at 340 nm.

5.5 Interpretation and Acceptance

1. System suitability: Standard curve must show r ≥ 0.980.
2. Assay validity: Recovery for positive product control (PPC) must be 50–200%.
3. Endotoxin limit: Based on dose and route, use calculations as per USP <85>.

5.6 Documentation

1. Record dilution factors, assay parameters, and raw OD values in Annexure-2.
2. Summarize results and interpretations in Annexure-3.
3. QA to review report and digitally sign validated results.

6. Abbreviations

• BET: Bacterial Endotoxin Test
• LAL: Limulus Amebocyte Lysate
• CSE: Control Standard Endotoxin
• EU: Endotoxin Unit
• PPC: Positive Product Control

7. Documents

1. Inhibition/Enhancement Test Record – Annexure-1
2. Raw Data and Optical Density Log – Annexure-2
3. Endotoxin Assay Summary Report – Annexure-3

8. References

• USP <85> Bacterial Endotoxins Test
• Ph. Eur. 2.6.14 Bacterial Endotoxins
• Indian Pharmacopoeia BET Chapter
• FDA Guidance for Industry: Pyrogen and Endotoxins Testing

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name	Manoj Jaiswal	Karishma Shah	Sunita Reddy
Designation	Microbiologist	QA Reviewer	Head – AMD
Department	Analytical Method Development	QA	Analytical Method Development

11. Annexures

Annexure-1: Inhibition/Enhancement Test Record

Sample	Dilution	PPC Recovery (%)	Status
WFI Lot 2405	1:10	110%	Pass

Annexure-2: Optical Density Log

Well No.	Sample ID	OD Reading (340 nm)	Time to Threshold (min)
A1	CSE 1.0 EU/mL	0.950	28

Annexure-3: Endotoxin Assay Summary

Sample ID	Endotoxin Result (EU/mL)	Limit	Status
INJ-2025-007	0.18	0.25	Pass

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
21/05/2025	2.0	Expanded to include OD-based calculations and software validation checkpoints	Annual Review	Sunita Reddy
10/06/2022	1.0	Initial SOP Release	New SOP	QA Head