Standard Operating Procedure for Transient Transfection Methods in Biosimilar R&D

Department	Biosimilars
SOP No.	SOP/BS/009/2025
Supersedes	SOP/BS/009/2022
Page No.	Page 1 of 13
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To define the procedure for transient transfection of mammalian cells using lipid-mediated or electroporation techniques for the expression of biosimilar proteins in early-stage R&D experiments.

2. Scope

This SOP applies to personnel involved in biosimilar R&D who perform gene delivery into cell lines like CHO, HEK293, or others for temporary expression analysis and protein production.

3. Responsibilities

• Research Scientist: Plans and executes the transfection experiment and analyzes protein expression.
• Lab Assistant: Prepares media, buffers, and ensures cell viability.
• QA Officer: Reviews transfection logs and performance documentation.

4. Accountability

The Head of Cell Culture and Expression Systems is accountable for ensuring the transfection protocols meet reproducibility and compliance standards.

5. Procedure

5.1 Cell Preparation

1. Culture cells (e.g., HEK293 or CHO) in appropriate media until they reach 70–90% confluency.
2. Use fresh, healthy cells between passages 5–20.
3. Count cells using a hemocytometer or cell counter and adjust to required density (e.g., 2 × 10⁵ cells/mL).

5.2 Lipid-Mediated Transfection Protocol

1. In a sterile tube, dilute 1 µg plasmid DNA in 50 µL of serum-free DMEM or Opti-MEM.
2. In a separate tube, dilute 2 µL of transfection reagent (e.g., Lipofectamine 3000) in 50 µL of the same medium.
3. Combine the two solutions, mix gently, and incubate at room temperature for 10–15 minutes.
4. Add the mixture dropwise to cells plated in a 24-well or 6-well plate.
5. Incubate at 37°C with 5% CO₂ for 24–72 hours.

5.3 Electroporation Protocol

1. Harvest 1 × 10⁶ cells and resuspend in 100 µL of electroporation buffer.
2. Add 2–5 µg of plasmid DNA to the cell suspension.
3. Transfer mixture into pre-chilled electroporation cuvette (2 mm gap).
4. Electroporate using program settings recommended for the specific cell type (e.g., 250 V, 950 µF).
5. Immediately transfer cells to recovery medium in a culture plate.

5.4 Expression Analysis

1. Harvest cells or media after 48–72 hours depending on the target protein.
2. Analyze expression using ELISA, SDS-PAGE, Western blot, or flow cytometry as applicable.
3. Record data in Transfection and Expression Log (Annexure-1).

5.5 Documentation and Storage

1. Label transfected samples with cell line, plasmid ID, and transfection date.
2. Store samples at -20°C or -80°C based on protein stability.
3. File all experiment sheets, instrument settings, and results in the R&D archive.

6. Abbreviations

• SOP: Standard Operating Procedure
• CHO: Chinese Hamster Ovary
• HEK: Human Embryonic Kidney
• ELISA: Enzyme-Linked Immunosorbent Assay
• DMEM: Dulbecco’s Modified Eagle Medium

7. Documents

1. Transfection and Expression Log (Annexure-1)
2. Cell Culture Condition Sheet (Annexure-2)

8. References

• ICH Q5D – Derivation and Characterization of Cell Substrates
• WHO Guidelines for Recombinant DNA Product Expression
• CDSCO Technical Guidelines on Cell-Based Expression Systems

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Transfection and Expression Log

Date	Cell Line	Plasmid ID	Method	Expression Time	Result	Operator
03/05/2025	HEK293	pCMV-rhEPO	Lipid	48 hr	Positive (Western Blot)	Rajesh Kumar

Annexure-2: Cell Culture Condition Sheet

Date	Cell Line	Media	Passage No.	Confluency	Used For
02/05/2025	CHO-K1	DMEM/F12 + 10% FBS	14	80%	Electroporation

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Included electroporation procedure and updated analysis techniques	Annual review update