Standard Operating Procedure for Protein Refolding (if applicable) in Biosimilar Manufacturing

Department	Biosimilars
SOP No.	SOP/BS/184/2025
Supersedes	SOP/BS/184/2022
Page No.	Page 1 of 10
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To establish a validated procedure for protein refolding of biosimilar drug substances expressed in bacterial systems as inclusion bodies, allowing recovery of biologically active conformation under GMP conditions.

2. Scope

This SOP applies to the downstream processing of biosimilar proteins requiring solubilization and refolding after extraction from inclusion bodies (IBs), typically in E. coli expression systems.

3. Responsibilities

• Production: Execute refolding process as per BMR, monitor parameters, and maintain records.
• Process Development: Provide optimized refolding buffer composition and time-temperature profile.
• QA: Ensure compliance with validation protocol and review all documentation.

4. Accountability

The Manufacturing Head is accountable for ensuring that protein refolding steps are executed under controlled and documented conditions according to GMP.

5. Procedure

5.1 Pre-Refolding Preparations

1. Ensure solubilized inclusion body protein is clarified by centrifugation and filtered using 0.45 µm filter.
2. Verify the protein concentration and denaturant concentration (e.g., 6–8 M urea or guanidine HCl).
3. Prepare refolding buffer as per BMR, maintaining pH, redox pair concentration (e.g., reduced and oxidized glutathione), and stabilizing agents.

5.2 Refolding by Dilution Method

1. Cool refolding buffer to 4–8°C if required to minimize aggregation.
2. Add solubilized protein dropwise (1:10 or 1:20 dilution ratio) into refolding buffer under constant stirring.
3. Maintain gentle stirring for 12–48 hours at predefined temperature (e.g., 10–25°C).
4. Monitor turbidity, optical density, and if applicable, sample for folding intermediates at defined intervals.

5.3 Refolding by Diafiltration Method (if applicable)

1. Load solubilized protein into UF/DF system equipped with 10–30 kDa MWCO membrane.
2. Exchange denaturant buffer with refolding buffer over 10–15 diavolumes.
3. Control TMP and flow rate to avoid protein denaturation during buffer exchange.

5.4 Post-Refolding Handling

1. After refolding, centrifuge at 10,000 × g for 30 min to remove precipitates.
2. Filter through 0.2 µm membrane and store at 2–8°C or proceed to purification step.

5.5 In-Process Controls

1. Collect samples for analytical assessment:
   • Protein concentration
   • Folding efficiency (e.g., CD, fluorescence, ELISA, HPLC)
   • Aggregation or turbidity checks

6. Abbreviations

• IB: Inclusion Body
• UF/DF: Ultrafiltration/Diafiltration
• CD: Circular Dichroism
• HPLC: High-Performance Liquid Chromatography

7. Documents

1. Refolding Monitoring Log – Annexure-1
2. Buffer Preparation Sheet – Annexure-2
3. Refolding Yield Record – Annexure-3

8. References

• ICH Q8 – Pharmaceutical Development
• WHO TRS 999 – GMP Guidelines for Biotherapeutics
• FDA Guidance – Development of Therapeutic Protein Products

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Refolding Monitoring Log

Time	Temp (°C)	OD @ 320nm	Sample Taken	Operator
08:00	10	0.030	Yes	Ajay Verma

Annexure-2: Buffer Preparation Sheet

Buffer Name	Components	pH	Storage Temp	Prepared By
Refolding Buffer A	Tris-HCl, GSH/GSSG	8.0	4°C	Sunita Reddy

Annexure-3: Refolding Yield Record

Batch ID	Initial Protein (mg)	Final Protein (mg)	Yield (%)	QC Verified
BS-IB-037	1050	882	84%	Yes

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Added diafiltration-based refolding and updated in-process controls	Process Validation