Standard Operating Procedure for Protein A Affinity Chromatography Setup in Biosimilar Manufacturing

Department	Biosimilars
SOP No.	SOP/BS/154/2025
Supersedes	SOP/BS/154/2022
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 GMP-compliant procedure for setting up Protein A affinity chromatography columns used in the capture and purification of monoclonal antibodies in biosimilar manufacturing processes.

2. Scope

This SOP applies to all single-use or reusable Protein A chromatography columns used in downstream processing (DSP) of biosimilar monoclonal antibodies at manufacturing scale.

3. Responsibilities

• Production: Perform setup, equilibration, and buffer line connections.
• QA: Verify batch record entries, review system readiness, and approve initiation of chromatography.
• Engineering: Maintain chromatography skid calibration and support troubleshooting.

4. Accountability

The Downstream Process Lead is accountable for ensuring that Protein A chromatography setup is performed as per validated process and recorded in GMP batch documentation.

5. Procedure

5.1 Pre-Setup Checks

1. Verify column ID, resin batch number, cleaning status, and integrity label.
2. Confirm column packing status (pre-packed vs in-house packed).
3. Record all pre-use checks in Annexure-1: Chromatography Setup Checklist.

5.2 Buffer Preparation and Line Assignment

1. Prepare and label the following buffers:
   • Equilibration Buffer (e.g., PBS, pH 7.4)
   • Binding Buffer
   • Elution Buffer (e.g., Glycine-HCl, pH 3.0)
   • Regeneration Buffer (e.g., 0.1 M NaOH)
   • Storage Buffer (e.g., 20% ethanol)
2. Assign buffer lines using color-coded, sanitized tubing.
3. Prime lines to remove air bubbles and avoid spiking during gradient runs.

5.3 Column Connection

1. Connect the column inlet and outlet to the skid using aseptic, leak-free connectors.
2. Ensure correct flow direction and backpressure tolerance (refer OEM specs).
3. Secure the column using appropriate clamp or housing base.

5.4 System Sanitization and Flow Path Integrity

1. Flush the column and associated skid path with 0.1 M NaOH for 20 minutes (if required).
2. Rinse with WFI until pH reaches 7.0 ± 0.2 and conductivity is below 10 µS/cm.
3. Check for leaks and record line integrity in Annexure-2: Flow Path Check Log.

5.5 Equilibration

1. Pass equilibration buffer at 100–150 cm/hr flow rate for 5–10 column volumes.
2. Monitor UV baseline stability and pH to ensure readiness for sample loading.

5.6 Documentation

1. Record:
   • Column ID and resin lot
   • Buffer names, lot numbers, and pH
   • Flow rate and pressure
   • Temperature (if controlled)
2. Attach skid-generated chromatogram to batch record.

6. Abbreviations

• PBS: Phosphate Buffered Saline
• DSP: Downstream Processing
• OEM: Original Equipment Manufacturer
• WFI: Water for Injection

7. Documents

1. Chromatography Setup Checklist – Annexure-1
2. Flow Path Check Log – Annexure-2

8. References

• WHO TRS 999 – Guidelines on Biotherapeutic Products
• ICH Q8 – Pharmaceutical Development
• OEM Manual – Cytiva/GE Healthcare Protein A Columns

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Chromatography Setup Checklist

Date	Column ID	Resin Batch	Pre-Use Sanitization	Leak Check	Operator
04/05/2025	PA-01	GE-RBT-2201	Yes	Pass	Ajay Verma

Annexure-2: Flow Path Check Log

Date	Skid ID	Buffer Line	Rinse pH	Conductivity	Status	Verified By
04/05/2025	CHM-120	A-EQ	7.1	8.5 µS/cm	OK	Sunita Reddy

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Updated sanitization and resin traceability steps	Process harmonization