Standard Operating Procedure for Hydrophobic Interaction Chromatography in Biosimilar Manufacturing

Department	Biosimilars
SOP No.	SOP/BS/163/2025
Supersedes	SOP/BS/163/2022
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 standard procedure for performing Hydrophobic Interaction Chromatography (HIC) for purification and polishing of biosimilar monoclonal antibodies or recombinant proteins under GMP-compliant conditions.

2. Scope

This SOP is applicable to downstream processing where HIC is used to remove aggregates, host cell proteins (HCP), and other hydrophobic impurities during biosimilar manufacturing.

3. Responsibilities

• Production: Setup, perform chromatography run, and document process parameters.
• QA: Review batch records and chromatographic data for compliance.
• Engineering: Maintain and calibrate chromatography skid and sensors.

4. Accountability

The Downstream Processing Lead is accountable for execution of HIC operations, ensuring process consistency, product integrity, and compliance with SOP.

5. Procedure

5.1 Materials and Buffer Preparation

1. Use HIC resin (e.g., Phenyl Sepharose, Butyl Sepharose) as per process development specifications.
2. Prepare the following buffers:
   • Equilibration Buffer: 20 mM phosphate, 1.5 M ammonium sulfate, pH 7.0
   • Elution Buffer: 20 mM phosphate, 0 M ammonium sulfate, pH 7.0
   • Cleaning Buffer: 0.5 M NaOH
   • Storage Buffer: 20% ethanol in WFI
3. Filter buffers using 0.22 µm filters and document in Annexure-1.

5.2 Column Equilibration

1. Install HIC column and ensure leak-free connections.
2. Equilibrate column with 5–10 column volumes (CV) of equilibration buffer at flow rate of 100 cm/hr.
3. Monitor UV (280 nm), conductivity, and pressure until baseline stabilizes.

5.3 Sample Preparation and Loading

1. Adjust product feed to high salt conditions using buffer exchange or stepwise addition of ammonium sulfate.
2. Filter the sample through a 0.22 µm filter before loading.
3. Load the sample at flow rate of 80–120 cm/hr and monitor UV absorbance.

5.4 Washing and Elution

1. Wash with equilibration buffer to remove unbound impurities.
2. Elute with decreasing salt gradient or direct application of elution buffer.
3. Collect fractions and monitor UV signal to identify product peak.
4. Pool fractions that meet acceptance criteria for purity and concentration.

5.5 Column Cleaning and Storage

1. Wash column with 0.5 M NaOH for 3 CV contact time.
2. Rinse with WFI until neutral pH is achieved.
3. Store column in 20% ethanol at 2–8°C.

5.6 Documentation

1. Record buffer lots, chromatography parameters, fraction volumes, and chromatograms.
2. Attach all analytical results (e.g., SDS-PAGE, SEC-HPLC) to batch record.

6. Abbreviations

• HIC: Hydrophobic Interaction Chromatography
• WFI: Water for Injection
• HCP: Host Cell Protein
• CV: Column Volume

7. Documents

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

8. References

• ICH Q8 – Pharmaceutical Development
• WHO TRS 999 – Biologic GMP Guidelines
• OEM Resin Manuals – Cytiva, Bio-Rad, Repligen

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	Salt Conc.	Volume	Filter ID	Prepared By	Date
Equilibration	7.0	1.5 M AmSO₄	10 L	FL-3124	Sunita Reddy	04/05/2025

Annexure-2: Chromatography Log Sheet

Step	Flow Rate	UV	Pressure	Start Time	End Time	Operator
Loading	100 cm/hr	0.85 AU	1.5 bar	11:00	11:45	Ajay Verma

Annexure-3: Cleaning and Storage Log

Date	Column ID	Cleaning Agent	Contact Time	Final pH	Storage Buffer	Stored At
04/05/2025	HIC-103	0.5 M NaOH	20 min	7.2	20% EtOH	2–8°C

Revision History:

Revision Date	Revision No.	Revision Details	Reason	Approved By
04/05/2025	2.0	Included SDS-PAGE and SEC analysis as standard evaluation	Process harmonization