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

1. Use pyrogen-free equipment, water (WFI), and buffers throughout the process.
2. Rinse all chromatography columns and containers with WFI prior to use.
3. Use dedicated tubing sets for bacterial expression systems to avoid cross-contamination.

5.2 Removal Techniques During Purification

1. 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.
2. Adsorptive Resins:
   • Use commercial endotoxin removal resins (e.g., Mustang Q, ETOPEARL) as an additional polishing step.
3. 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

1. Collect samples after each major purification step and analyze for endotoxin using LAL assay.
2. Document results in Annexure-1.

5.4 Acceptance Criteria

1. 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

1. All product contact surfaces must be part of validated cleaning protocols with rinse and swab recovery studies.
2. 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

1. Endotoxin Test Log – Annexure-1
2. 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