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
- Verify the resin type and column ID. Record resin lot and column pre-use status in Annexure-1.
- 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
- Document buffer preparation parameters in Annexure-2.
5.2 Column Equilibration
- Mount column vertically and connect tubing correctly (ensure flow is top-to-bottom or per resin manufacturer).
- Equilibrate with 5–10 column volumes (CV) of equilibration buffer.
- Monitor and record UV (280 nm), conductivity, and pressure.
5.3 Sample Loading
- Ensure that the sample is filtered through 0.22 µm and is pH-adjusted to match equilibration buffer.
- Load sample at 100–200 cm/hr, maintaining pressure < 2 bar.
- Observe UV for breakthrough and confirm loading endpoint.
5.4 Washing and Elution
- Wash with 5 CV of equilibration buffer to remove unbound materials.
- Elute with NaCl gradient (e.g., 0–500 mM over 10 CV) or step elution.
- Collect fractions and analyze UV absorbance and conductivity to pool peak product fractions.
5.5 Column Cleaning and Storage
- Clean column using 0.5 M NaOH for 20–30 minutes at room temperature.
- Flush with WFI until neutral pH is reached.
- Store in 20% ethanol at 2–8°C if not used immediately.
5.6 Documentation
- Attach printouts of UV and conductivity profiles to batch records.
- Record all chromatography parameters in Annexure-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
- Column ID and Resin Log – Annexure-1
- Buffer Preparation Record – Annexure-2
- Chromatography Run Log – Annexure-3
- 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 |