Standard Operating Procedure for Sanitization of Chromatography Columns in Biosimilar Manufacturing

Department	Biosimilars
SOP No.	SOP/BS/166/2025
Supersedes	SOP/BS/166/2022
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 sanitizing chromatography columns used in the purification of biosimilar proteins to prevent microbial contamination, ensure resin longevity, and maintain GMP compliance.

2. Scope

This SOP applies to all chromatography columns (pre-packed or manually packed) used in downstream processing, including ion-exchange, hydrophobic interaction, affinity, and size exclusion chromatography systems.

3. Responsibilities

• Production: Perform sanitization using approved reagents and ensure completion of all steps as per protocol.
• QA: Review sanitization records, chemical usage logs, and approve column for subsequent runs.
• Engineering: Maintain cleaning skids and pumps used during sanitization procedures.

4. Accountability

The Downstream Production Manager is accountable for proper sanitization, execution documentation, and ensuring cleaning verification is completed.

5. Procedure

5.1 Preparation

1. Ensure the column is properly connected to a validated cleaning-in-place (CIP) skid or chromatography system.
2. Verify that all hoses and seals are intact and leak-free.
3. Prepare the sanitizing agent as per validated concentration (e.g., 0.5 M NaOH, 0.1 M NaOH, or 2% NaOCl).
4. Confirm availability of WFI for rinsing and storage buffer for column preservation.

5.2 Sanitization Process

1. Flush the column with WFI for 1–2 column volumes (CV) to remove residual product or buffers.
2. Introduce sanitizing agent at a flow rate of 50–100 cm/hr.
3. Allow static contact for minimum 30 minutes to achieve bioburden reduction.
4. Recirculate sanitizing agent if specified by resin manufacturer (e.g., 3 cycles).

5.3 Post-Sanitization Rinsing

1. Flush column with WFI until pH of effluent matches that of WFI (typically 6.0–7.0).
2. Monitor conductivity to ensure removal of sanitizing agent (target < 10 µS/cm).
3. Document number of rinse cycles and volume used in Annexure-1.

5.4 Storage

1. If the column is not immediately reused, flush with storage buffer (e.g., 20% ethanol in WFI).
2. Seal both ends with sanitary plugs and label with date of sanitization and next due date.
3. Store horizontally (for large columns) or as per resin manufacturer’s recommendation.

5.5 Cleaning Verification

1. Conduct rinse sampling for TOC (Total Organic Carbon) and residual NaOH verification.
2. Ensure all acceptance criteria are met before releasing column for production use.
3. Record verification data in Annexure-2.

6. Abbreviations

• CIP: Clean-In-Place
• WFI: Water for Injection
• TOC: Total Organic Carbon
• CV: Column Volume

7. Documents

1. Sanitization Record – Annexure-1
2. Cleaning Verification Report – Annexure-2

8. References

• ICH Q9 – Quality Risk Management
• WHO TRS 999 – GMP Guidelines for Biotherapeutics
• OEM Resin Cleaning Guidelines (GE/Cytiva, Repligen, Bio-Rad)

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Sanitization Record

Date	Column ID	Sanitizing Agent	Contact Time	Rinse Cycles	Operator
04/05/2025	COL-HIC-104	0.5 M NaOH	30 min	3	Ajay Verma

Annexure-2: Cleaning Verification Report

Parameter	Limit	Result	Pass/Fail	Verified By
pH of final rinse	6.0–7.5	6.8	Pass	Sunita Reddy
Conductivity	< 10 µS/cm	6.2 µS/cm	Pass	Sunita Reddy
TOC	< 500 ppb	410 ppb	Pass	Sunita Reddy

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Included TOC and conductivity verification for final rinse	QA audit recommendation