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

Department	Biosimilars
SOP No.	SOP/BS/171/2025
Supersedes	SOP/BS/171/2022
Page No.	Page 1 of 9
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To define the validated procedure for removing residual host cell DNA during biosimilar purification using chromatography and enzymatic digestion methods to meet regulatory thresholds for DNA content in the final drug substance.

2. Scope

This SOP applies to biosimilar drug substances expressed in mammalian (e.g., CHO) or bacterial (e.g., E. coli) systems where host cell DNA must be reduced to acceptable limits as per global regulatory guidelines.

3. Responsibilities

• Production: Execute enzymatic treatment and monitor downstream steps for DNA clearance.
• QC: Perform residual DNA testing (e.g., qPCR or PicoGreen assay).
• QA: Review test reports and ensure all steps align with validated parameters.

4. Accountability

The Downstream Process Lead is accountable for successful implementation of DNA removal strategy and maintenance of product quality and compliance.

5. Procedure

5.1 Identification of DNA Removal Steps

1. Nuclease Treatment:
   • Use Benzonase or other endonuclease during early purification stage.
   • Add enzyme post-harvest in buffer containing Mg²⁺ (1–2 mM) at 25–37°C for 30–60 min.
2. Capture Chromatography:
   • Protein A or IEX capture removes significant proportion of host DNA.
   • Optimize wash steps to ensure displacement of loosely bound DNA.
3. Polishing Chromatography:
   • AEX or HIC further removes trace DNA fragments via charge-based exclusion.

5.2 Enzyme Inactivation

1. Inactivate nuclease post-treatment using validated methods such as:
   • pH shift to >10.0 for 15 minutes
   • Buffer exchange by TFF to remove enzyme and breakdown products

5.3 Sample Testing and Monitoring

1. Collect samples at post-nuclease, post-capture, and final drug substance stage.
2. Analyze using:
   • qPCR: DNA quantitation down to pg/mL levels
   • PicoGreen Assay: For fluorescent detection of double-stranded DNA
3. Record test results in Annexure-1.

5.4 Acceptance Criteria

1. Final drug substance must contain <10 ng DNA per dose (WHO and FDA guidelines).
2. Results must be within validated batch range.

5.5 Documentation and Compliance

1. Document all DNA removal steps in batch records with timestamps and volumes.
2. Attach DNA test reports with analyst signatures and QA verification.

6. Abbreviations

• DNA: Deoxyribonucleic Acid
• AEX: Anion Exchange Chromatography
• TFF: Tangential Flow Filtration
• qPCR: Quantitative Polymerase Chain Reaction
• HIC: Hydrophobic Interaction Chromatography

7. Documents

1. DNA Clearance Log – Annexure-1
2. qPCR Assay Report – Annexure-2

8. References

• ICH Q6B – Test Procedures and Acceptance Criteria for Biotechnological Products
• WHO TRS 987 – DNA Residual Guidelines
• FDA Guidance for Industry – Residual DNA in Biologics

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: DNA Clearance Log

Sample Point	Sample ID	DNA Concentration (ng/mL)	Test Method	Analyst	Date
Post-Nuclease	DNA-01	32	qPCR	Ajay Verma	04/05/2025
Post-Polishing	DNA-02	5.4	PicoGreen	Ajay Verma	04/05/2025

Annexure-2: qPCR Assay Report

Run ID	DNA Standard Curve R²	LOD (ng/mL)	Sample Result	Pass/Fail
QP-2025-14	0.998	0.5	5.4	Pass

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Included enzyme inactivation and qPCR testing protocol	Regulatory harmonization