Standard Operating Procedure for Western Blotting for Protein Expression in Biosimilar Cell Clones
| Department | Biosimilars |
|---|---|
| SOP No. | SOP/BS/014/2025 |
| Supersedes | SOP/BS/014/2022 |
| Page No. | Page 1 of 12 |
| Issue Date | 04/05/2025 |
| Effective Date | 06/05/2025 |
| Review Date | 04/05/2026 |
1. Purpose
To define the standardized procedure for detecting and analyzing biosimilar protein expression in cell lysates using SDS-PAGE followed by Western blotting during clone evaluation and selection.
2. Scope
This SOP is applicable to research and analytical staff involved in screening and confirming protein expression in cell-based biosimilar production systems using immunoblotting techniques.
3. Responsibilities
- Analytical Scientist: Performs protein extraction, SDS-PAGE, blotting, and detection.
- Lab Technician: Assists in reagent preparation and operation of blotting apparatus.
- QA Officer: Verifies blotting data and ensures procedural documentation is complete.
4. Accountability
The Head of Analytical Development is accountable for ensuring the Western blotting process is performed accurately and results are validated and recorded per regulatory guidelines.
5. Procedure
5.1 Sample Preparation
- Harvest cells and wash with PBS.
- Lyse cells using RIPA buffer supplemented with protease inhibitors.
- Centrifuge at 13,000 rpm for 15 minutes at 4°C and collect supernatant.
- Quantify protein concentration using BCA or Bradford assay.
5.2 SDS-PAGE Electrophoresis
- Prepare 10–12% polyacrylamide gels depending on target protein size.
- Mix equal volumes of sample (20–40 µg protein) with 2X Laemmli buffer and heat at 95°C for 5 minutes.
- Load molecular weight marker and samples into gel wells.
- Run gel at 100–120V until dye front reaches the bottom.
5.3 Transfer to Membrane
- Assemble gel and PVDF/nitrocellulose membrane in blotting cassette.
- Transfer proteins at 100V for 1 hour or 30V overnight at 4°C.
- Confirm transfer by Ponceau S staining if required.
5.4 Blocking and Antibody Incubation
- Block membrane with 5% skim milk or BSA in TBST for 1 hour at room temperature.
- Incubate with primary antibody (e.g., anti-biosimilar protein) in blocking buffer for 2 hours or overnight at 4°C.
- Wash membrane 3× with TBST, 5 minutes each wash.
- Incubate with HRP-conjugated secondary antibody for 1 hour at room temperature.
- Repeat washing step post-secondary antibody incubation.
5.5 Detection and Documentation
- Develop blot using chemiluminescent substrate.
- Capture signal using gel documentation system or X-ray film.
- Record exposure time, band intensity, and molecular weight in Western Blot Analysis Log (Annexure-1).
6. Abbreviations
- SDS-PAGE: Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
- HRP: Horseradish Peroxidase
- TBST: Tris Buffered Saline with Tween 20
- BSA: Bovine Serum Albumin
7. Documents
- Western Blot Analysis Log (Annexure-1)
- Antibody and Reagent Inventory Sheet (Annexure-2)
8. References
- ICH Q6B – Specifications: Test Procedures for Biotechnological Products
- WHO Guidelines on Biosimilar Characterization
- CDSCO Analytical Method Validation Guidance
9. SOP Version
Version: 2.0
10. Approval Section
| Prepared By | Checked By | Approved By | |
|---|---|---|---|
| Signature | |||
| Date | |||
| Name | |||
| Designation | |||
| Department |
11. Annexures
Annexure-1: Western Blot Analysis Log
| Date | Clone ID | Protein Target | Band Size (kDa) | Intensity | Remarks | Operator |
|---|---|---|---|---|---|---|
| 03/05/2025 | CL-BS-014 | rhEPO | 34 | Strong | Expected size observed | Sunita Reddy |
Annexure-2: Antibody and Reagent Inventory Sheet
| Item | Lot No. | Expiry | Storage | Stock Qty |
|---|---|---|---|---|
| Anti-rhEPO Antibody | AB2345 | 10/2026 | 2–8°C | 3 vials |
| HRP-Secondary | HRP7832 | 12/2025 | 2–8°C | 5 vials |
Revision History:
| Revision Date | Revision No. | Revision Details | Reason for Revision | Approved By |
|---|---|---|---|---|
| 04/05/2025 | 2.0 | Added documentation templates and optimized blocking step | Annual review |