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
- 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
- Document method selected in the BMR and align with process development data.
5.2 Chromatography-Based Endotoxin Removal
- Equilibrate column with low ionic strength buffer (e.g., 20 mM Tris, pH 8.0).
- Load clarified protein solution at ≤2 mg/mL concentration.
- Collect flow-through fractions and test for protein yield and endotoxin level.
- Pool only fractions with endotoxin level ≤0.1 EU/mg protein.
5.3 Phase Separation (if applicable)
- Add Triton X-114 (final 1% v/v) to protein solution and incubate at 4°C for 30 min.
- Warm to 37°C for 15 min and centrifuge at 5,000 × g for 15 minutes.
- Recover aqueous phase carefully and discard detergent-rich lower phase.
5.4 Ultrafiltration Option
- Use 100–300 kDa MWCO membrane to separate protein from endotoxin aggregates.
- Diafilter with low conductivity buffer for 10–15 diavolumes.
5.5 In-Process Control and Testing
- QC to test all critical process fractions using LAL kinetic turbidimetric or chromogenic assay.
- 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
- Endotoxin Reduction Log – Annexure-1
- Buffer Preparation Sheet – Annexure-2
- 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 |