Standard Operating Procedure for Protein Expression Profiling in Biosimilar-Producing Cell Lines

Department	Biosimilars
SOP No.	SOP/BS/020/2025
Supersedes	SOP/BS/020/2022
Page No.	Page 1 of 14
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To define the procedure for evaluating biosimilar protein expression levels in engineered cell clones using SDS-PAGE, Western blotting, and densitometry techniques for clone selection and development decisions.

2. Scope

This SOP is applicable to all personnel in the Biosimilars department responsible for assessing recombinant protein expression levels in stably transfected or transiently expressing mammalian cell lines.

3. Responsibilities

• Research Scientist: Designs experimental layout and reviews results.
• Lab Technician: Carries out protein extraction, electrophoresis, blotting, and quantification.
• QA Officer: Ensures records are maintained and verifies consistency with GMP documentation practices.

4. Accountability

The Head of Analytical Development is accountable for the accuracy and regulatory compliance of protein expression data used for biosimilar development and clone selection.

5. Procedure

5.1 Sample Collection and Protein Extraction

1. Harvest 1–2 × 10⁶ cells by centrifugation at 300 × g for 5 min.
2. Wash cell pellet with ice-cold PBS.
3. Lyse cells in RIPA buffer containing protease inhibitors.
4. Incubate on ice for 30 minutes and centrifuge at 12,000 × g for 15 minutes.
5. Collect supernatant and measure protein concentration using BCA assay.

5.2 SDS-PAGE

1. Load 20–40 µg of total protein per lane on a 10–12% SDS-PAGE gel.
2. Run at 120V until dye front reaches bottom of gel.

5.3 Western Blotting

1. Transfer proteins to PVDF membrane at 100V for 90 minutes in transfer buffer.
2. Block membrane in 5% non-fat milk in TBST for 1 hour at room temperature.
3. Incubate with primary antibody (e.g., anti-rhEPO) overnight at 4°C.
4. Wash and incubate with HRP-conjugated secondary antibody for 1 hour at room temperature.
5. Detect signals using chemiluminescent substrate and capture on X-ray film or imaging system.

5.4 Densitometry and Data Analysis

1. Scan membrane and quantify band intensity using image analysis software (e.g., ImageJ).
2. Normalize expression to housekeeping protein (e.g., β-actin).
3. Document results in Expression Profiling Log (Annexure-1).

6. Abbreviations

• SDS-PAGE: Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
• HRP: Horseradish Peroxidase
• BCA: Bicinchoninic Acid
• PVDF: Polyvinylidene Fluoride

7. Documents

1. Expression Profiling Log (Annexure-1)
2. Protein Quantification Log (Annexure-2)

8. References

• ICH Q6B – Specifications: Test Procedures and Acceptance Criteria
• WHO Guidelines for Biosimilar Characterization
• MIQE Guidelines – Western Blotting Best Practices

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Expression Profiling Log

Date	Clone ID	Band Size (kDa)	Intensity (AU)	Normalized Expression	Operator
03/05/2025	CL-BS-020	34	1520	1.25	Rajesh Kumar

Annexure-2: Protein Quantification Log

Date	Sample ID	Protein Concentration (µg/µL)	Method	Remarks	Operator
02/05/2025	CL-BS-020-Lysate	2.4	BCA	Good yield	Sunita Reddy

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Expanded procedure with densitometry and SDS-PAGE controls	Annual SOP review