Biosimilars: SOP for Protein Expression Profiling – V 2.0
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
- Harvest 1–2 × 106 cells by centrifugation at 300 × g for 5 min.
- Wash cell pellet with ice-cold PBS.
- Lyse cells in RIPA buffer containing protease inhibitors.
- Incubate on ice for 30 minutes and centrifuge at 12,000 × g for 15 minutes.
- Collect supernatant and measure protein concentration using BCA assay.
5.2 SDS-PAGE
- Load 20–40 µg of total protein per lane on a 10–12% SDS-PAGE gel.
- Run at 120V until dye front reaches bottom of gel.
5.3 Western Blotting
- Transfer proteins to PVDF membrane at 100V for 90 minutes in transfer buffer.
- Block membrane in 5% non-fat milk in TBST for 1 hour at room temperature.
- Incubate with primary antibody (e.g., anti-rhEPO) overnight at 4°C.
- Wash and incubate with HRP-conjugated secondary antibody for 1 hour at room temperature.
- Detect signals using chemiluminescent substrate and capture on X-ray film or imaging system.
5.4 Densitometry and Data Analysis
- Scan membrane and quantify band intensity using image analysis software (e.g., ImageJ).
- Normalize expression to housekeeping protein (e.g., β-actin).
- 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
- Expression Profiling Log (Annexure-1)
- 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
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Checked By |
Approved By |
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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 |
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