Standard Operating Procedure for Removal of Host Cell Proteins in Biosimilar Manufacturing

Department	Biosimilars
SOP No.	SOP/BS/170/2025
Supersedes	SOP/BS/170/2022
Page No.	Page 1 of 10
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To outline the procedure for effective removal of host cell proteins (HCP) during the downstream processing of biosimilars using validated purification steps to ensure product purity and compliance with GMP requirements.

2. Scope

This SOP applies to all biosimilar drug substances expressed in prokaryotic or mammalian systems (e.g., CHO cells, E. coli) where HCP clearance is critical during purification and polishing processes.

3. Responsibilities

• Production: Execute the purification and polishing steps that contribute to HCP removal.
• QC: Perform ELISA-based HCP testing on in-process and final samples.
• QA: Review all batch records and HCP clearance data for release authorization.

4. Accountability

The Downstream Process Manager is accountable for ensuring all purification steps are implemented as per validated process and are effective in clearing HCP contaminants.

5. Procedure

5.1 Critical Points of HCP Removal

1. Capture Step (Protein A or Ion-Exchange Chromatography):
   • Remove bulk of HCP through selective binding of target protein.
   • Optimize binding and wash buffers to displace weakly bound impurities.
2. Intermediate Purification (IEX or HIC):
   • Use salt gradients or hydrophobic interactions to separate co-eluting HCPs.
   • Collect fractions based on UV signal and predefined retention volumes.
3. Polishing Step (SEC or AEX):
   • Apply size-based or charge-based separation to further purify the product.
   • Ensure final HCP levels are below specification (e.g., < 100 ppm).

5.2 Sample Collection and Testing

1. Collect samples after each chromatography step.
2. Submit for HCP testing using platform-specific ELISA (CHO-HCP or E. coli-HCP kits).
3. Document all test results in Annexure-1.

5.3 Process Monitoring and Optimization

1. Use historical trend charts to compare HCP clearance efficiency.
2. Revise buffer conditions or flow rates if HCP levels deviate from acceptable limits.
3. Maintain records of each change and rationale in Annexure-2.

5.4 Documentation and Compliance

1. Ensure chromatography logbooks reflect HCP-relevant details (column ID, buffers, gradient conditions).
2. Attach HCP test certificates to BMR (Batch Manufacturing Record).

6. Abbreviations

• HCP: Host Cell Protein
• ELISA: Enzyme-Linked Immunosorbent Assay
• SEC: Size Exclusion Chromatography
• IEX: Ion Exchange Chromatography
• AEX: Anion Exchange Chromatography
• BMR: Batch Manufacturing Record

7. Documents

1. HCP Test Result Log – Annexure-1
2. HCP Process Deviation and Correction Log – Annexure-2

8. References

• ICH Q6B – Specifications: Test Procedures and Acceptance Criteria for Biotechnological Products
• WHO TRS 999 – GMP for Biotherapeutics
• FDA Guidance – Immunogenicity Testing of Therapeutic Protein 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: HCP Test Result Log

Step	Sample ID	HCP (ppm)	Acceptance Criteria	Analyst	Date
Capture	BS-HCP-2025-01	600	<1000	Sunita Reddy	04/05/2025
Intermediate	BS-HCP-2025-02	150	<500	Sunita Reddy	04/05/2025
Polishing	BS-HCP-2025-03	22	<100	Sunita Reddy	04/05/2025

Annexure-2: HCP Process Deviation and Correction Log

Date	Deviation Observed	Corrective Action	Performed By	Approved By
03/05/2025	Elevated HCP post-IEX	Increased wash CV by 50%	Ajay Verma	QA Manager

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Updated polishing conditions and acceptance limits for new ELISA kit	Analytical platform upgrade