Standard Operating Procedure for Viral Inactivation via Low pH Treatment in Biosimilar Manufacturing

Department	Biosimilars
SOP No.	SOP/BS/157/2025
Supersedes	SOP/BS/157/2022
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 procedure for performing viral inactivation through low pH treatment of the product intermediate during biosimilar monoclonal antibody purification, ensuring safety and compliance with regulatory viral clearance expectations.

2. Scope

This SOP applies to low pH viral inactivation steps performed post-elution and pre-intermediate purification for biosimilar antibody drug substances at the downstream processing stage.

3. Responsibilities

• Production: Perform pH adjustment, hold monitoring, and documentation.
• QA: Review inactivation log and validate hold time conditions.
• QC: Perform pH verification and sampling for post-treatment testing.

4. Accountability

The Downstream Process Lead is accountable for proper execution of viral inactivation and recording critical control parameters under validated conditions.

5. Procedure

5.1 Pre-Checks and Preparation

1. Ensure all previous purification steps (e.g., Protein A elution) are completed.
2. Prepare acidifying agent (e.g., 1 M acetic acid or HCl) and neutralization buffer (e.g., Tris-HCl, pH 9.0).
3. Ensure calibrated pH meter is available for verification and pH probes are sanitized.
4. Record batch number, product volume, and starting pH in Annexure-1: Inactivation Log Sheet.

5.2 pH Adjustment for Inactivation

1. Transfer eluted product into viral inactivation vessel under controlled conditions (Grade C area minimum).
2. Start slow mixing and gradually add acidifying agent until solution pH reaches validated target (typically pH 3.5 ± 0.1).
3. Verify pH with calibrated pH meter at multiple points in the vessel to ensure homogeneity.
4. Start the viral inactivation hold time once target pH is confirmed and maintained.

5.3 Inactivation Hold Time

1. Maintain continuous mixing throughout the hold period (typically 60 minutes ± validated window).
2. Document:
   • Start and end time
   • pH every 15 minutes
   • Room temperature (if applicable)
3. Any deviation from validated pH range or time window must be reported immediately to QA.

5.4 Post-Inactivation Neutralization

1. After the validated hold time is completed, adjust pH back to neutral range (pH 7.0 ± 0.2) using Tris-HCl buffer.
2. Mix thoroughly and confirm final pH using pH meter.
3. Record post-neutralization pH and temperature in Annexure-2: pH Adjustment Record.

5.5 Sample Collection and Disposal

1. Collect samples for:
   • Bioburden
   • Viral clearance (if required)
   • Protein concentration and pH verification
2. Dispose of acid and base handling containers as per biohazard SOP.

6. Abbreviations

• pH: Potential of Hydrogen
• QA: Quality Assurance
• QC: Quality Control
• AU: Absorbance Units

7. Documents

1. Inactivation Log Sheet – Annexure-1
2. pH Adjustment Record – Annexure-2

8. References

• ICH Q5A(R1) – Viral Safety Evaluation of Biotechnology Products
• WHO TRS 978 – GMP for Biotherapeutic Products
• Product-specific Viral Clearance Validation Protocol

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Inactivation Log Sheet

Date	Batch No.	Start Time	End Time	Initial pH	Final pH	Operator
04/05/2025	BS-DP-085	12:30	13:30	3.4	3.5	Ajay Verma

Annexure-2: pH Adjustment Record

Time	pH Reading	Action Taken	Remarks	Recorded By
13:45	6.9	Added Tris-HCl	Achieved pH target	Sunita Reddy

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Expanded hold time monitoring and post-neutralization steps	Regulatory alignment