Standard Operating Procedure for Strong Cation Exchange Chromatography in Biosimilar Manufacturing

Department	Biosimilars
SOP No.	SOP/BS/160/2025
Supersedes	SOP/BS/160/2022
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 performing strong cation exchange (SCX) chromatography for intermediate or polishing purification of biosimilar monoclonal antibodies, enabling removal of host cell proteins, aggregates, and charge variants under GMP guidelines.

2. Scope

This SOP is applicable to the use of SCX resins such as SP Sepharose, Fractogel EMD SO₃⁻, and equivalent materials for purification during downstream processing (DSP) of biosimilar products.

3. Responsibilities

• Production: Execute column operations, monitor parameters, and maintain documentation.
• QA: Review operation logs and buffer preparations.
• Engineering: Maintain chromatography skid and support troubleshooting.

4. Accountability

The Downstream Processing Lead is accountable for process adherence, equipment readiness, and data integrity during SCX operations.

5. Procedure

5.1 Column and Buffer Preparation

1. Verify that the SCX column (e.g., SP Sepharose) is properly packed or pre-packed and labeled.
2. Prepare the following buffers:
   • Equilibration Buffer: e.g., 20 mM sodium acetate, pH 5.0
   • Elution Buffer: same as equilibration buffer with 0–500 mM NaCl gradient
   • Cleaning Buffer: 0.5 M NaOH
   • Storage Buffer: 20% ethanol
3. Filter and document all buffer preparations in Annexure-1.

5.2 Column Equilibration

1. Install the column on the skid and secure connections.
2. Equilibrate the column with 5–10 column volumes of equilibration buffer at a flow rate of 100–200 cm/hr.
3. Monitor UV (280 nm), conductivity, and pressure throughout the process.

5.3 Sample Loading

1. Ensure sample pH is within 0.2 units of the equilibration buffer and has low conductivity.
2. Load sample at an optimized flow rate (e.g., 100–150 cm/hr) based on column dimensions.
3. Record UV peak height and monitor pressure differentials.

5.4 Washing and Elution

1. Wash with equilibration buffer to remove unbound components.
2. Elute using a linear or stepwise NaCl gradient over 10 column volumes.
3. Collect fractions based on UV signal or defined conductivity windows.
4. Analyze fractions for protein content, pH, and purity.

5.5 Cleaning and Storage

1. Clean column with 0.5 M NaOH for 3–5 column volumes.
2. Rinse with WFI until neutral pH is achieved.
3. Store column in 20% ethanol at 2–8°C if not reused immediately.

5.6 Documentation

1. Record all chromatography parameters, buffer lots, and sample details in Annexures 1–3.
2. Attach chromatograms to the BMR and submit for QA review.

6. Abbreviations

• SCX: Strong Cation Exchange
• DSP: Downstream Processing
• NaOH: Sodium Hydroxide
• WFI: Water for Injection

7. Documents

1. Buffer Preparation Record – Annexure-1
2. SCX Chromatography Operation Log – Annexure-2
3. Cleaning and Storage Log – Annexure-3

8. References

• ICH Q8 – Pharmaceutical Development
• WHO TRS 999 – GMP Guidelines for Biologics
• OEM Chromatography Resin Manuals (e.g., Cytiva, 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: Buffer Preparation Record

Buffer Name	pH	Conductivity	Prepared By	Date	Filter ID
Elution Buffer	5.0	38 mS/cm	Sunita Reddy	04/05/2025	FL-2047

Annexure-2: SCX Chromatography Operation Log

Step	Flow Rate	UV	Pressure	Start Time	End Time	Operator
Sample Load	125 cm/hr	0.68 AU	1.3 bar	12:00	12:40	Ajay Verma

Annexure-3: Cleaning and Storage Log

Date	Column ID	Cleaning Agent	Contact Time	Final pH	Stored In
04/05/2025	SCX-002	0.5 M NaOH	30 min	7.2	20% ethanol

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Updated UV and pressure monitoring, added cleaning contact time	Process validation alignment