Standard Operating Procedure for Intermediate Purification via Ion-Exchange Chromatography in Biosimilar Manufacturing

Department	Biosimilars
SOP No.	SOP/BS/159/2025
Supersedes	SOP/BS/159/2022
Page No.	Page 1 of 11
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To describe the procedure for performing intermediate purification of biosimilar monoclonal antibodies using ion-exchange chromatography (IEX), ensuring removal of process impurities under GMP-compliant conditions.

2. Scope

This SOP is applicable to the use of both cation and anion exchange chromatography steps in downstream processing of biosimilar therapeutic proteins using pre-packed or in-house packed columns.

3. Responsibilities

• Production: Set up IEX system, perform equilibration, sample loading, and elution.
• QA: Review all batch records, IEX logs, and monitor compliance.
• Engineering: Ensure skid, pump, and sensor calibration and maintenance.

4. Accountability

The DSP Lead is accountable for ensuring proper execution of intermediate purification using IEX and for overseeing all documentation.

5. Procedure

5.1 Column and Buffer Preparation

1. Select appropriate resin (e.g., Q-Sepharose, SP Sepharose) based on process validation.
2. Prepare and filter all required buffers:
   • Equilibration Buffer
   • Sample Buffer (pH-adjusted)
   • Wash Buffer (low salt)
   • Elution Buffer (NaCl gradient or stepwise)
   • Regeneration and Storage Buffers
3. Record buffer pH, conductivity, and lot numbers in Annexure-1.

5.2 Column Setup and Equilibration

1. Install column securely on the chromatography skid.
2. Connect inlet and outlet tubing and verify correct direction of flow.
3. Prime and equilibrate the column with 5–10 column volumes of equilibration buffer.
4. Monitor UV, pH, and conductivity until stable baseline is achieved.

5.3 Sample Loading

1. Transfer clarified and filtered feed to the loading tank.
2. Set flow rate to 100–200 cm/hr based on column size and resin type.
3. Monitor column pressure and UV absorbance at 280 nm during loading.
4. Terminate loading when breakthrough is detected or sample is exhausted.

5.4 Washing and Elution

1. Wash column with 3–5 CV of wash buffer to remove unbound impurities.
2. Elute target protein using:
   • Linear gradient of NaCl (e.g., 0–500 mM over 10 CV), or
   • Step elution (predefined NaCl concentration)
3. Monitor UV and collect fractions where product peak elutes.

5.5 Column Regeneration

1. Wash column with high salt or alkaline buffer (e.g., 0.5 M NaOH) for 3–5 CV.
2. Rinse thoroughly with WFI or storage buffer.
3. Store column in 20% ethanol at 2–8°C if not reused immediately.

5.6 Documentation

1. Record:
   • Column ID and resin lot
   • Buffer volumes and flow rates
   • UV chromatogram and collection timepoints
   • Any deviation or alerts triggered during run
2. Complete Annexures 1 to 3 as part of the BMR.

6. Abbreviations

• IEX: Ion Exchange Chromatography
• CV: Column Volume
• UV: Ultraviolet Absorbance
• DSP: Downstream Processing

7. Documents

1. Buffer Preparation Record – Annexure-1
2. Chromatography Operation Log – Annexure-2
3. Column Regeneration and Storage Log – Annexure-3

8. References

• ICH Q8 – Pharmaceutical Development
• WHO TRS 999 – GMP for Biotherapeutics
• OEM Manuals – Cytiva IEX Columns, Repligen, Sartorius Systems

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Buffer Preparation Record

Buffer	pH	Conductivity	Volume	Prepared By	Date
Elution Buffer	7.5	42 mS/cm	10 L	Sunita Reddy	04/05/2025

Annexure-2: Chromatography Operation Log

Step	Flow Rate	Pressure	UV (280nm)	Start Time	End Time	Operator
Loading	125 cm/hr	1.2 bar	0.6 AU	10:00	10:45	Ajay Verma

Annexure-3: Column Regeneration and Storage Log

Date	Column ID	Regeneration Solution	Contact Time	Storage Buffer	Stored At
04/05/2025	IEX-Q-01	0.5 M NaOH	30 min	20% ethanol	2–8°C

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Added buffer conductivity control and UV breakthrough monitoring	Process harmonization