Standard Operating Procedure for Strong Anion Exchange Chromatography in Biosimilar Manufacturing

Department	Biosimilars
SOP No.	SOP/BS/161/2025
Supersedes	SOP/BS/161/2022
Page No.	Page 1 of 11
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To establish a standard procedure for conducting strong anion exchange (AEX) chromatography for intermediate purification or polishing of biosimilar proteins by removing process-related impurities, host cell DNA, and viral particles.

2. Scope

This SOP is applicable to the use of strong AEX chromatography resins such as Q Sepharose, DEAE resins, or equivalent for downstream processing of biosimilar monoclonal antibodies or other recombinant proteins.

3. Responsibilities

• Production: Perform column setup, equilibration, sample loading, elution, and cleaning.
• QA: Review batch records, buffer logs, and chromatography process parameters.
• Engineering: Ensure skid and sensor calibration and maintenance readiness.

4. Accountability

The Downstream Manufacturing Head is accountable for process adherence and completeness of documentation associated with AEX operations.

5. Procedure

5.1 Preparation

1. Verify the resin type and column ID. Record resin lot and column pre-use status in Annexure-1.
2. Prepare and filter buffers as per validated process:
   • Equilibration Buffer: 20 mM Tris-HCl, pH 8.0
   • Elution Buffer: same as equilibration buffer with 0–500 mM NaCl
   • Cleaning Buffer: 0.5 M NaOH
   • Storage Buffer: 20% ethanol in WFI
3. Document buffer preparation parameters in Annexure-2.

5.2 Column Equilibration

1. Mount column vertically and connect tubing correctly (ensure flow is top-to-bottom or per resin manufacturer).
2. Equilibrate with 5–10 column volumes (CV) of equilibration buffer.
3. Monitor and record UV (280 nm), conductivity, and pressure.

5.3 Sample Loading

1. Ensure that the sample is filtered through 0.22 µm and is pH-adjusted to match equilibration buffer.
2. Load sample at 100–200 cm/hr, maintaining pressure < 2 bar.
3. Observe UV for breakthrough and confirm loading endpoint.

5.4 Washing and Elution

1. Wash with 5 CV of equilibration buffer to remove unbound materials.
2. Elute with NaCl gradient (e.g., 0–500 mM over 10 CV) or step elution.
3. Collect fractions and analyze UV absorbance and conductivity to pool peak product fractions.

5.5 Column Cleaning and Storage

1. Clean column using 0.5 M NaOH for 20–30 minutes at room temperature.
2. Flush with WFI until neutral pH is reached.
3. Store in 20% ethanol at 2–8°C if not used immediately.

5.6 Documentation

1. Attach printouts of UV and conductivity profiles to batch records.
2. Record all chromatography parameters in Annexure-3.
3. Complete cleaning and storage log in Annexure-4.

6. Abbreviations

• AEX: Anion Exchange Chromatography
• CV: Column Volume
• WFI: Water for Injection
• NaCl: Sodium Chloride

7. Documents

1. Column ID and Resin Log – Annexure-1
2. Buffer Preparation Record – Annexure-2
3. Chromatography Run Log – Annexure-3
4. Cleaning and Storage Record – Annexure-4

8. References

• ICH Q8 – Pharmaceutical Development
• WHO TRS 999 – GMP for Biotherapeutics
• OEM Guidelines – Cytiva, Repligen, Sartorius resin manuals

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Column ID and Resin Log

Date	Column ID	Resin Type	Lot No.	Status	Verified By
04/05/2025	AEX-Q-01	Q Sepharose	QSP-01234	Ready	Ajay Verma

Annexure-2: Buffer Preparation Record

Buffer	pH	Conductivity	Prepared By	Date	Filter ID
Elution Buffer	8.0	45 mS/cm	Sunita Reddy	04/05/2025	FL-2450

Annexure-3: Chromatography Run Log

Step	Flow Rate	UV	Pressure	Start Time	End Time	Operator
Sample Load	150 cm/hr	0.72 AU	1.4 bar	11:30	12:10	Ajay Verma

Annexure-4: Cleaning and Storage Record

Date	Column ID	Cleaning Agent	Contact Time	Final pH	Storage Conditions
04/05/2025	AEX-Q-01	0.5 M NaOH	30 min	7.1	20% Ethanol @ 2–8°C

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Updated resin types and added hold time for cleaning	Regulatory update