Standard Operating Procedure for Elution and Regeneration of Protein A Column in Biosimilar Manufacturing

Department	Biosimilars
SOP No.	SOP/BS/156/2025
Supersedes	SOP/BS/156/2022
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 the procedure for elution of bound monoclonal antibodies from Protein A affinity chromatography columns and subsequent regeneration of the column for reuse in biosimilar downstream processing.

2. Scope

This SOP is applicable to all Protein A columns used in the purification of monoclonal antibodies (mAbs) in biosimilar manufacturing across single-use or reusable chromatography systems.

3. Responsibilities

• Production: Execute elution and regeneration procedures and monitor critical parameters.
• QA: Verify and review elution recovery logs and resin regeneration data.
• Engineering: Maintain and calibrate chromatography skids and monitor pressure profiles.

4. Accountability

The Head of Downstream Processing is accountable for ensuring successful product recovery and column reuse under validated and GMP-compliant conditions.

5. Procedure

5.1 Elution of Monoclonal Antibody

1. Ensure equilibration and product loading are completed as per SOP/BS/155/2025.
2. Prepare Elution Buffer (e.g., 0.1 M Glycine-HCl, pH 2.8–3.0).
3. Connect the elution buffer line to the chromatography skid.
4. Set the elution parameters:
   • Flow rate: 80–100 cm/hr
   • Volume: 3–5 column volumes (CV) or as validated
5. Monitor UV at 280 nm. Start elution collection when UV absorbance exceeds 0.05 AU.
6. Collect elution peak in pre-labeled sterile bags or bottles.
7. Neutralize pooled elution fractions immediately using neutralization buffer (e.g., 1 M Tris-HCl, pH 8.0).
8. Record the elution profile in Annexure-1: Elution Log Sheet and attach chromatogram printouts.

5.2 Post-Elution Wash

1. Flush column with equilibration buffer until baseline UV is restored.
2. This step removes loosely bound contaminants before regeneration.

5.3 Column Regeneration

1. Prepare Regeneration Buffer (e.g., 0.1–0.5 M NaOH).
2. Pass regeneration buffer at 100–150 cm/hr for 3–5 CV or for 20 minutes, whichever is longer.
3. Allow static contact (soaking) for 15–30 minutes if specified in the resin manufacturer’s protocol.
4. Rinse with WFI or storage buffer until pH stabilizes at 6.8–7.2.
5. Record resin contact time and buffer volumes in Annexure-2: Regeneration Record.

5.4 Resin Storage (Reusable Columns)

1. Store resin in appropriate storage solution (e.g., 20% ethanol) at 2–8°C if not reused immediately.
2. Label storage status clearly on column tag and log storage start time.

5.5 Disposal (Single-Use Columns)

1. Disconnect and discard single-use columns per biohazard disposal SOPs.
2. Document lot traceability in Annexure-3: Column Usage and Disposal Log.

6. Abbreviations

• mAb: Monoclonal Antibody
• CV: Column Volume
• NaOH: Sodium Hydroxide
• WFI: Water for Injection

7. Documents

1. Elution Log Sheet – Annexure-1
2. Regeneration Record – Annexure-2
3. Column Usage and Disposal Log – Annexure-3

8. References

• WHO TRS 999 – Guidelines on Biotherapeutic Products
• ICH Q8 – Pharmaceutical Development
• OEM Resin Manual – Cytiva MabSelect, Repligen Amsphere

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Elution Log Sheet

Date	Batch No.	Elution Buffer	Start Time	End Time	Volume Collected	Operator
04/05/2025	BS-DP-084	Gly-HCl, pH 3.0	11:00	11:45	2.2 L	Ajay Verma

Annexure-2: Regeneration Record

Date	Column ID	NaOH Conc.	Contact Time	Rinse Volume	Final pH	Performed By
04/05/2025	PA-01	0.5 M	20 min	8 L	7.1	Sunita Reddy

Annexure-3: Column Usage and Disposal Log

Column ID	Used In Batch	No. of Cycles	Storage/Disposal	Action Date	Handled By
PA-SU-202	BS-DP-084	1	Disposed	04/05/2025	QA Officer

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Updated resin storage and disposal steps for single-use columns	Compliance update