Standard Operating Procedure for Endotoxin Testing in Cell Culture for Biosimilars

Department	Biosimilars
SOP No.	SOP/BS/031/2025
Supersedes	SOP/BS/031/2022
Page No.	Page 1 of 12
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To define a validated procedure for testing endotoxin levels in biosimilar cell culture media, buffers, and harvested fluids using the Limulus Amebocyte Lysate (LAL) assay to ensure product safety and compliance.

2. Scope

This SOP applies to the QC Microbiology department involved in biosimilar production processes where monitoring of endotoxin levels is critical, including raw materials, in-process samples, and cell harvests.

3. Responsibilities

• QC Analyst: Prepares samples, performs LAL assay, and documents test results.
• QC Supervisor: Reviews assay performance, evaluates interference, and signs reports.
• QA Officer: Ensures LAL kits, standards, and results meet GMP documentation practices.

4. Accountability

The Head of Quality Control is accountable for ensuring that endotoxin testing is performed according to pharmacopeial standards and that results are valid and reviewed prior to lot release.

5. Procedure

5.1 Preparation

1. Ensure the use of depyrogenated glassware and LAL reagent water (LRW).
2. Allow LAL reagents and control standard endotoxin (CSE) to equilibrate to room temperature.
3. Prepare standard curve using CSE in LRW (e.g., 0.25 EU/mL to 1.0 EU/mL).

5.2 Sample Collection

1. Collect 1–2 mL of sample in depyrogenated vials using aseptic technique.
2. Label with sample ID, date, matrix (e.g., culture media, harvest fluid).
3. If dilution is required, use LRW only.

5.3 Gel Clot Method (Qualitative)

1. Mix equal volumes (100 µL each) of LAL reagent and sample/standard/control.
2. Incubate tubes at 37 ±1°C for 60 ±2 minutes undisturbed.
3. Invert tubes gently. If clot remains intact → Positive; no clot → Negative.

5.4 Kinetic Chromogenic Method (Quantitative)

1. Add 100 µL of sample and 100 µL of chromogenic LAL reagent into microplate wells.
2. Run the plate in a kinetic LAL reader and record absorbance at 405 nm.
3. Generate endotoxin concentration from standard curve.

5.5 Interference Testing

1. Conduct spike recovery test:
   • Add known CSE to sample
   • Recover within 50–200% to validate sample matrix
2. If recovery fails, dilute sample or use alternate method.

5.6 Acceptance Criteria

1. Endotoxin limits as per USP <85> or ICH Q6B or as specified in product specifications (e.g., <0.5 EU/mL).
2. All controls (positive, negative, spike) must pass for results to be valid.

5.7 Documentation

1. Record results in Endotoxin Test Log (Annexure-1).
2. Prepare Endotoxin Test Report (Annexure-2) and forward to QA for review.

6. Abbreviations

• LAL: Limulus Amebocyte Lysate
• LRW: LAL Reagent Water
• CSE: Control Standard Endotoxin
• EU: Endotoxin Units

7. Documents

1. Endotoxin Test Log (Annexure-1)
2. Endotoxin Test Report (Annexure-2)

8. References

• USP <85> – Bacterial Endotoxins Test
• ICH Q6B – Specifications: Biological Products
• WHO TRS 999 – GMP for Biological Products

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 Test Log

Date	Sample ID	Method	Result (EU/mL)	Analyst	Remarks
03/05/2025	BS-HF-031	Kinetic Chromogenic	0.23	Sunita Reddy	Within limit

Annexure-2: Endotoxin Test Report

Sample ID	Matrix	Method	Date Tested	Endotoxin Result	Limit	Approved By
BS-HF-031	Harvest Fluid	Kinetic	03/05/2025	0.23 EU/mL	0.5 EU/mL

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Integrated kinetic method and added matrix interference check	Regulatory update