Standard Operating Procedure for Polishing Step Using Size Exclusion Chromatography in Biosimilar Manufacturing

Department	Biosimilars
SOP No.	SOP/BS/164/2025
Supersedes	SOP/BS/164/2022
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 polishing purification of biosimilar products using size exclusion chromatography (SEC) to remove aggregates and achieve final product purity under GMP conditions.

2. Scope

This SOP applies to downstream processing where SEC is utilized as the final polishing step for biosimilar monoclonal antibodies or recombinant proteins in biopharmaceutical manufacturing.

3. Responsibilities

• Production: Execute SEC operations, monitor UV and flow parameters, and complete documentation.
• QA: Review SEC logs, batch records, and chromatograms for compliance.
• Engineering: Ensure equipment calibration and proper operation of chromatography skids.

4. Accountability

The DSP Supervisor is accountable for the correct execution of SEC runs, data integrity, and reporting deviations during operation.

5. Procedure

5.1 Column and Buffer Preparation

1. Use pre-packed or in-house packed SEC columns (e.g., Superdex 200 Increase, Sephacryl S-300).
2. Prepare isocratic buffer:
   • 20 mM sodium phosphate, 150 mM NaCl, pH 7.0
3. Filter buffer through 0.22 µm filter and record in Annexure-1.

5.2 Column Equilibration

1. Equilibrate column with 1.5–2 column volumes (CV) of isocratic buffer.
2. Flow rate must not exceed manufacturer’s recommended linear velocity (e.g., 15 cm/hr).
3. Monitor baseline UV at 280 nm before sample injection.

5.3 Sample Preparation and Injection

1. Filter sample through 0.22 µm membrane before loading.
2. Sample volume should not exceed 2–5% of column volume to maintain resolution.
3. Inject sample and monitor real-time UV signal.

5.4 Elution and Fraction Collection

1. Use isocratic elution (no gradient).
2. Monitor and record UV absorbance for product, monomer, and aggregate peaks.
3. Collect fractions corresponding to the monomer peak only.
4. Analyze by SEC-HPLC to confirm aggregate removal.

5.5 Post-Run Cleaning and Storage

1. Flush column with at least 2 CV of WFI.
2. If reused: sanitize with 0.1 M NaOH and re-equilibrate.
3. Store column in 20% ethanol at 2–8°C.

5.6 Documentation

1. Complete logs for buffer preparation, column ID, injection volumes, and chromatography data.
2. Attach chromatograms to batch record for QA review.

6. Abbreviations

• SEC: Size Exclusion Chromatography
• DSP: Downstream Processing
• CV: Column Volume
• WFI: Water for Injection

7. Documents

1. Buffer Preparation Record – Annexure-1
2. SEC Chromatography Log Sheet – Annexure-2
3. Cleaning and Storage Record – Annexure-3

8. References

• ICH Q8 – Pharmaceutical Development
• WHO TRS 999 – GMP Guidelines for Biologics
• OEM Manuals – Cytiva Superdex, Tosoh Bioscience TSKgel

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
SEC Buffer	7.0	14.8 mS/cm	10 L	Sunita Reddy	04/05/2025

Annexure-2: SEC Chromatography Log Sheet

Sample ID	Injection Volume	Flow Rate	Run Start	Run End	Operator
BS-POL-062	10 mL	0.3 mL/min	12:10	13:00	Ajay Verma

Annexure-3: Cleaning and Storage Record

Date	Column ID	Cleaning Agent	Storage Buffer	Storage Temp.	Operator
04/05/2025	SEC-103	0.1 M NaOH	20% EtOH	2–8°C	Ajay Verma

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Added SEC-HPLC confirmation and clarified sample injection limits	Polishing step optimization