Standard Operating Procedure for Equilibration and Loading of Protein A Column in Biosimilar Manufacturing

Department	Biosimilars
SOP No.	SOP/BS/155/2025
Supersedes	SOP/BS/155/2022
Page No.	Page 1 of 10
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To describe the procedure for equilibrating and loading the Protein A chromatography column used for capture purification of monoclonal antibodies (mAbs) in biosimilar manufacturing under GMP conditions.

2. Scope

This SOP applies to all manufacturing batches involving Protein A capture chromatography using pre-packed or in-house packed columns in downstream processing facilities.

3. Responsibilities

• Production: Execute equilibration and loading, monitor parameters, and record results.
• QA: Review batch records and approve process execution.
• Engineering: Ensure skid and pressure sensor functionality prior to use.

4. Accountability

The Downstream Production Manager is accountable for adherence to validated Protein A loading protocols and ensuring GMP compliance in documentation.

5. Procedure

5.1 Pre-Equilibration Checks

1. Ensure the column has been properly sanitized and flushed as per SOP/BS/154/2025.
2. Confirm the availability of equilibration buffer and that it has been filtered and labeled.
3. Record buffer lot number, pH, and conductivity in Annexure-1: Equilibration and Loading Log.

5.2 Column Equilibration

1. Connect the equilibration buffer line to the chromatography skid inlet.
2. Prime the line and eliminate all air pockets.
3. Set skid parameters:
   • Flow rate: 100–150 cm/hr
   • Volume: 5–10 column volumes (CV)
4. Start buffer flow and monitor UV (280 nm), pH, and conductivity.
5. Stabilize baseline (UV < 0.05 AU) before starting sample loading.

5.3 Loading of Product

1. Connect harvest tank or depth-filtered clarified harvest to the skid product inlet.
2. Ensure the harvest has passed pre-use bioburden and turbidity checks.
3. Set skid:
   • Loading flow rate: 100–120 cm/hr
   • UV load limit: As defined in process validation or BMR (e.g., 70% DBC)
4. Monitor:
   • Back pressure (must not exceed resin limit)
   • UV absorbance (to detect breakthrough)
5. Terminate loading once breakthrough is detected or volume threshold is reached.

5.4 Column Wash (Optional)

1. Pass 3–5 CV of wash buffer to remove weakly bound impurities (if part of validated protocol).
2. Collect flowthrough in designated containers for analysis or discard.

5.5 Data Recording

1. Record:
   • Buffer and sample volumes
   • Start/end times
   • Skid run ID
   • UV trend and batch comments
2. Attach chromatogram printout and submit for QA review.

6. Abbreviations

• CV: Column Volume
• UV: Ultraviolet
• DBC: Dynamic Binding Capacity
• mAb: Monoclonal Antibody

7. Documents

1. Equilibration and Loading Log – Annexure-1
2. Buffer Preparation Record – Annexure-2

8. References

• ICH Q8 – Pharmaceutical Development
• WHO TRS 999 – Guidelines on Biotherapeutic Products
• Protein A OEM Technical Manual – Cytiva / Repligen

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Equilibration and Loading Log

Date	Batch No.	Buffer Lot	Flow Rate	UV at Load	Column ID	Operator
04/05/2025	BS-DP-083	PBS-BL-05	125 cm/hr	0.54 AU	PA-01	Ajay Verma

Annexure-2: Buffer Preparation Record

Buffer Name	pH	Conductivity	Prepared By	Lot No.	Date
Equilibration Buffer	7.2	14.2 mS/cm	Sunita Reddy	PBS-BL-05	03/05/2025

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Added UV monitoring step and breakthrough handling	Operational improvement