Standard Operating Procedure for Endotoxin Removal in Bacterial Systems in Biosimilar Manufacturing

Department	Biosimilars
SOP No.	SOP/BS/187/2025
Supersedes	SOP/BS/187/2022
Page No.	Page 1 of 10
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To define a validated and GMP-compliant procedure for the effective removal of bacterial endotoxins (lipopolysaccharides, LPS) from biosimilar proteins expressed in E. coli to ensure product safety and regulatory compliance.

2. Scope

This SOP is applicable to all biosimilar drug substances derived from E. coli expression systems where endotoxin contamination must be reduced below pharmacopeial limits prior to downstream purification, formulation, or bulk release.

3. Responsibilities

• Production: Perform endotoxin removal steps as per BMR using validated resins or filters.
• QC: Conduct endotoxin testing using LAL assay and verify in-process reduction levels.
• QA: Review batch records and approve compliance with predefined endotoxin acceptance limits.

4. Accountability

The Head of Downstream Processing is accountable for ensuring complete and traceable removal of endotoxins in alignment with process validation and release specifications.

5. Procedure

5.1 Selection of Removal Strategy

1. Choose suitable removal method based on protein characteristics:
   • Anion exchange chromatography (AEX)
   • Polymyxin B affinity resin
   • Triton X-114 phase separation
   • Ultrafiltration with size exclusion
2. Document method selected in the BMR and align with process development data.

5.2 Chromatography-Based Endotoxin Removal

1. Equilibrate column with low ionic strength buffer (e.g., 20 mM Tris, pH 8.0).
2. Load clarified protein solution at ≤2 mg/mL concentration.
3. Collect flow-through fractions and test for protein yield and endotoxin level.
4. Pool only fractions with endotoxin level ≤0.1 EU/mg protein.

5.3 Phase Separation (if applicable)

1. Add Triton X-114 (final 1% v/v) to protein solution and incubate at 4°C for 30 min.
2. Warm to 37°C for 15 min and centrifuge at 5,000 × g for 15 minutes.
3. Recover aqueous phase carefully and discard detergent-rich lower phase.

5.4 Ultrafiltration Option

1. Use 100–300 kDa MWCO membrane to separate protein from endotoxin aggregates.
2. Diafilter with low conductivity buffer for 10–15 diavolumes.

5.5 In-Process Control and Testing

1. QC to test all critical process fractions using LAL kinetic turbidimetric or chromogenic assay.
2. Ensure final endotoxin content is ≤0.1 EU/mg of protein or as per product specification.

6. Abbreviations

• AEX: Anion Exchange Chromatography
• LAL: Limulus Amebocyte Lysate
• MWCO: Molecular Weight Cut-Off
• EU: Endotoxin Unit

7. Documents

1. Endotoxin Reduction Log – Annexure-1
2. Buffer Preparation Sheet – Annexure-2
3. Endotoxin Test Record – Annexure-3

8. References

• USP <85> Bacterial Endotoxins Test
• ICH Q6B – Specifications for Biotechnological Products
• WHO TRS 999 – GMP Guidelines for Biotherapeutics

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 Reduction Log

Date	Batch No.	Method Used	Pre-Removal EU/mg	Post-Removal EU/mg	Operator
04/05/2025	BS-ENDO-040	Polymyxin B Resin	2.3	0.07	Ajay Verma

Annexure-2: Buffer Preparation Sheet

Buffer Name	Components	pH	Prepared By
AEX Buffer A	20 mM Tris-HCl	8.0	Sunita Reddy

Annexure-3: Endotoxin Test Record

Sample ID	Test Method	Result (EU/mg)	QC Analyst	Date
FT-05	LAL Kinetic	0.07	Dr. Reema Nair	04/05/2025

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Added alternate removal methods and clarified acceptance criteria	GMP alignment and method harmonization