Standard Operating Procedure for Reduction of Endotoxin Levels in Biosimilar Manufacturing
| Department | Biosimilars |
|---|---|
| SOP No. | SOP/BS/172/2025 |
| Supersedes | SOP/BS/172/2022 |
| Page No. | Page 1 of 10 |
| Issue Date | 04/05/2025 |
| Effective Date | 06/05/2025 |
| Review Date | 04/05/2026 |
1. Purpose
To establish a validated procedure for reducing endotoxin levels in biosimilar manufacturing using filtration, chromatography, and cleaning controls to ensure product safety and regulatory compliance.
2. Scope
This SOP applies to all purification processes involving bacterial or mammalian cell expression systems in biosimilar production where endotoxin removal is critical for parenteral drug product development.
3. Responsibilities
- Production: Execute all purification and cleaning operations designed to minimize endotoxin contamination.
- QC: Perform endotoxin testing using LAL (Limulus Amebocyte Lysate) assay on in-process and final bulk samples.
- QA: Review results, deviation records, and ensure compliance with validated endotoxin clearance procedures.
4. Accountability
The DSP Supervisor is accountable for maintaining acceptable endotoxin levels and adherence to defined cleaning and filtration protocols.
5. Procedure
5.1 Prevention of Endotoxin Introduction
- Use pyrogen-free equipment, water (WFI), and buffers throughout the process.
- Rinse all chromatography columns and containers with WFI prior to use.
- Use dedicated tubing sets for bacterial expression systems to avoid cross-contamination.
5.2 Removal Techniques During Purification
- Anion Exchange Chromatography (AEX):
- Endotoxins are negatively charged at neutral pH and can be separated using AEX in flow-through mode.
- Optimize buffer pH and conductivity to retain target protein while endotoxins flow through or bind to resin.
- Adsorptive Resins:
- Use commercial endotoxin removal resins (e.g., Mustang Q, ETOPEARL) as an additional polishing step.
- Ultrafiltration/Diafiltration (UF/DF):
- Implement TFF systems with 10–100 kDa MWCO membranes to reduce low molecular weight contaminants including endotoxins.
- Perform 5–10 diavolumes of diafiltration with WFI-equivalent buffer.
5.3 In-Process Testing
- Collect samples after each major purification step and analyze for endotoxin using LAL assay.
- Document results in Annexure-1.
5.4 Acceptance Criteria
- Endotoxin levels must not exceed:
- Parenteral products: 5 EU/kg/hr or <0.25 EU/mL (per USP/EP)
5.5 Cleaning Validation and Hold Time Study
- All product contact surfaces must be part of validated cleaning protocols with rinse and swab recovery studies.
- Hold time for buffers and intermediate pools must not exceed validated durations to prevent endotoxin growth.
6. Abbreviations
- EU: Endotoxin Units
- LAL: Limulus Amebocyte Lysate
- WFI: Water for Injection
- AEX: Anion Exchange Chromatography
- UF/DF: Ultrafiltration/Diafiltration
7. Documents
- Endotoxin Test Log – Annexure-1
- Cleaning Validation Summary – Annexure-2
8. References
- USP <85> Bacterial Endotoxins Test
- ICH Q6B – Specifications for Biotech Products
- FDA Guidance – Pyrogen and Endotoxin Testing
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
| Sample Point | Sample ID | Endotoxin Level (EU/mL) | Limit | Analyst | Date |
|---|---|---|---|---|---|
| Post-AEX | ETX-2025-01 | 0.18 | 0.25 | Ajay Verma | 04/05/2025 |
| Final Bulk | ETX-2025-02 | 0.04 | 0.25 | Ajay Verma | 04/05/2025 |
Annexure-2: Cleaning Validation Summary
| Equipment ID | Surface | Swab Result (EU/cm²) | Rinse Result (EU/mL) | Status |
|---|---|---|---|---|
| DSP-Tank-05 | Inner wall | 0.01 | 0.03 | Pass |
Revision History:
| Revision Date | Revision No. | Details | Reason | Approved By |
|---|---|---|---|---|
| 04/05/2025 | 2.0 | Added UF/DF and AEX resin specification for endotoxin removal | Process improvement |