Standard Operating Procedure for Inclusion Body Solubilization (if expressed in E. coli) in Biosimilar Manufacturing

Department	Biosimilars
SOP No.	SOP/BS/186/2025
Supersedes	SOP/BS/186/2022
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 GMP-compliant procedure for the solubilization of inclusion bodies (IBs) expressed in E. coli during biosimilar protein production, enabling downstream purification or refolding.

2. Scope

This SOP applies to all biosimilar products expressed as insoluble aggregates in bacterial systems (E. coli) and requiring solubilization using chaotropic or reducing agents under controlled processing conditions.

3. Responsibilities

• Production: Perform solubilization, maintain processing parameters, and document the batch data.
• Process Development: Define solubilization buffer composition and mixing strategy.
• QA: Ensure that all materials and processes conform to GMP and batch record specifications.

4. Accountability

The Manufacturing Manager is accountable for ensuring accurate execution, monitoring, and documentation of inclusion body solubilization operations.

5. Procedure

5.1 Preparation

1. Ensure inclusion bodies have been isolated from cell pellets via lysis and centrifugation steps.
2. Verify availability of solubilization buffer (e.g., 6M guanidine HCl or 8M urea) and reducing agents (e.g., 10 mM DTT).
3. Pre-chill or equilibrate processing vessel if temperature-sensitive operations are needed.

5.2 Solubilization Process

1. Weigh the IB pellet and suspend in solubilization buffer at a 1:10 (w/v) ratio.
2. Use overhead stirrer or magnetic agitation at 100–300 rpm for 2–4 hours at 20–25°C.
3. Ensure complete dissolution by visual inspection or reduction in turbidity (OD600).
4. If high viscosity is observed, dilute with additional solubilization buffer and continue stirring.

5.3 Clarification

1. Centrifuge solubilized IB solution at 10,000 × g for 30 minutes to remove insoluble debris.
2. Filter the supernatant using 0.45 µm PES membrane filter.
3. Collect clarified solubilized protein into a pre-cleaned, labeled container.

5.4 In-Process Testing

1. Collect sample and perform:
   • Protein concentration (UV280, BCA assay)
   • Solubility visual check
   • pH and conductivity measurement

5.5 Storage or Further Processing

1. Use solubilized IB for immediate downstream processing (e.g., refolding) or store at 2–8°C for a defined period.
2. Label with product ID, date, and operator initials.

6. Abbreviations

• IB: Inclusion Body
• DTT: Dithiothreitol
• PES: Polyethersulfone
• OD: Optical Density

7. Documents

1. IB Solubilization Log – Annexure-1
2. Solubilization Buffer Preparation Sheet – Annexure-2
3. Clarification and Filtration Record – Annexure-3

8. References

• ICH Q5C – Stability Testing of Biotechnological/Biological Products
• WHO TRS 999 – GMP for Biotherapeutics
• FDA Guidance for Industry – Quality Systems Approach to Pharmaceutical CGMP Regulations

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: IB Solubilization Log

Date	Batch No.	IB Weight (g)	Buffer Volume (mL)	Stir Time (hrs)	Operator
04/05/2025	BS-IB-038	60	600	3	Ajay Verma

Annexure-2: Solubilization Buffer Preparation Sheet

Component	Quantity	Final Concentration	pH	Prepared By
Guanidine HCl	57 g	6 M	8.0	Sunita Reddy

Annexure-3: Clarification and Filtration Record

Time	Centrifuge RPM	Temp (°C)	Filter Used	Volume Recovered (mL)	Remarks
12:00	10,000	20	0.45 µm PES	570	Clear, no particulates

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Added in-process testing steps and clarified agitation parameters	Process Validation Alignment