Standard Operating Procedure for Removal of Aggregates and Fragments in Biosimilar Manufacturing

Department	Biosimilars
SOP No.	SOP/BS/173/2025
Supersedes	SOP/BS/173/2022
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 procedure for the removal of aggregates and fragments from biosimilar protein products during downstream processing, ensuring high monomer purity and compliance with regulatory specifications.

2. Scope

This SOP applies to monoclonal antibodies and recombinant proteins expressed in biosimilar production platforms that require removal of high molecular weight (HMW) aggregates and low molecular weight (LMW) fragments during the polishing phase.

3. Responsibilities

• Production: Execute polishing steps using validated chromatography to separate aggregates/fragments.
• QC: Test in-process and final bulk samples using SEC-HPLC or CE-SDS to quantify monomer and impurities.
• QA: Ensure all activities and results are recorded and comply with GMP and product release specifications.

4. Accountability

The Head of Downstream Processing is accountable for ensuring that purification operations yield biosimilar proteins within acceptable purity limits and aggregate specifications.

5. Procedure

5.1 Identification of Critical Steps

1. Size Exclusion Chromatography (SEC):
   • Used as the primary method to remove aggregates and fragments post-AEX or HIC polishing.
   • Calibrate SEC columns with molecular weight markers to identify elution windows for monomer, aggregates, and fragments.
2. Preparative SEC Parameters:
   • Column: Superdex 200 pg or equivalent
   • Buffer: 20 mM phosphate, 150 mM NaCl, pH 7.0
   • Flow rate: 0.5–1.0 CV/hour
3. Collect monomer-containing fractions and discard early (aggregates) and late (fragments) eluting peaks.

5.2 Alternative Methods for Aggregate Removal

1. Anion Exchange in Flow-Through Mode:
   • Adjust conductivity and pH to bind impurities while monomer flows through.
2. Hydrophobic Interaction Chromatography:
   • Use gradient elution to achieve differential retention of misfolded aggregates.

5.3 In-Process Testing and Monitoring

1. Analyze each pool using:
   • SEC-HPLC: Monomer purity should be >98%
   • CE-SDS (Non-reducing): Detect fragments and dimer forms
2. Document test data in Annexure-1.

5.4 Acceptance Criteria

1. Aggregates: <2% HMW forms by SEC-HPLC
2. Fragments: <1% LMW species
3. Monomer Purity: >98% required for release

5.5 Documentation

1. Maintain chromatography run logs, pooling records, and test results in the batch manufacturing record.
2. QA to verify that only fractions meeting criteria are released for formulation.

6. Abbreviations

• SEC: Size Exclusion Chromatography
• HMW: High Molecular Weight (Aggregates)
• LMW: Low Molecular Weight (Fragments)
• CV: Column Volume
• CE-SDS: Capillary Electrophoresis-Sodium Dodecyl Sulfate

7. Documents

1. SEC Purity Test Report – Annexure-1
2. Pooling and Rejection Log – Annexure-2

8. References

• ICH Q6B – Test Procedures and Acceptance Criteria for Biotech Products
• WHO TRS 999 – GMP for Biotherapeutic Products
• FDA Guidance – Immunogenicity Testing and Aggregate Characterization

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: SEC Purity Test Report

Sample ID	Monomer (%)	Aggregate (%)	Fragment (%)	Result	Analyst
BS-FNL-SEC-001	98.6	1.2	0.2	Pass	Ajay Verma

Annexure-2: Pooling and Rejection Log

Fraction ID	Retention Time (min)	Content	Pooled (Y/N)	Remarks
F1	5.0	Aggregates	No	Rejected
F2	6.2	Monomer	Yes	Pooled
F3	7.8	Fragments	No	Discarded

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Integrated SEC and CE-SDS test data with new pooling criteria	Process optimization