SOP Guide for Pharma

Biosimilars: SOP for Western Blotting for Protein Expression – V 2.0

Auditor


Biosimilars: SOP for Western Blotting for Protein Expression – V 2.0


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

See also  Biosimilars: SOP for Restriction Enzyme Digestion and Mapping - V 2.0

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

  1. Harvest cells and wash with PBS.
  2. Lyse cells using RIPA buffer supplemented with protease inhibitors.
  3. Centrifuge at 13,000 rpm for 15 minutes at 4°C and collect supernatant.
  4. Quantify protein concentration using BCA or Bradford assay.

5.2 SDS-PAGE Electrophoresis

  1. Prepare 10–12% polyacrylamide gels depending on target protein size.
  2. Mix equal volumes of sample (20–40 µg protein) with 2X Laemmli buffer and heat at 95°C for 5 minutes.
  3. Load molecular weight marker and samples into gel wells.
  4. Run gel at 100–120V until dye front reaches the bottom.

5.3 Transfer to Membrane

  1. Assemble gel and PVDF/nitrocellulose membrane in blotting cassette.
  2. Transfer proteins at 100V for 1 hour or 30V overnight at 4°C.
  3. Confirm transfer by Ponceau S staining if required.
See also  Biosimilars: SOP for Glycosylation Analysis in Clone Selection - V 2.0

5.4 Blocking and Antibody Incubation

  1. Block membrane with 5% skim milk or BSA in TBST for 1 hour at room temperature.
  2. Incubate with primary antibody (e.g., anti-biosimilar protein) in blocking buffer for 2 hours or overnight at 4°C.
  3. Wash membrane 3× with TBST, 5 minutes each wash.
  4. Incubate with HRP-conjugated secondary antibody for 1 hour at room temperature.
  5. Repeat washing step post-secondary antibody incubation.

5.5 Detection and Documentation

  1. Develop blot using chemiluminescent substrate.
  2. Capture signal using gel documentation system or X-ray film.
  3. Record exposure time, band intensity, and molecular weight in Western Blot Analysis Log (Annexure-1).

6. Abbreviations

7. Documents

  1. Western Blot Analysis Log (Annexure-1)
  2. Antibody and Reagent Inventory Sheet (Annexure-2)
See also  Biosimilars: SOP for Productivity Testing in Small-Scale Cultures - V 2.0

8. References

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
Exit mobile version