Standard Operating Procedure for Troubleshooting Transfection Failures in Biosimilar Cell Line Development

Department	Biosimilars
SOP No.	SOP/BS/055/2025
Supersedes	SOP/BS/055/2022
Page No.	Page 1 of 11
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To establish a systematic approach for identifying and resolving transfection failures encountered during biosimilar cell line development to ensure optimal gene delivery efficiency and reproducibility.

2. Scope

This SOP applies to all transient and stable transfection experiments involving mammalian cell lines such as CHO and HEK293 conducted under the biosimilar development program.

3. Responsibilities

• Research Associates: Monitor and document transfection efficiency; initiate troubleshooting steps.
• CLD Scientists: Evaluate failure root causes and recommend experimental modifications.
• QA: Review deviations and verify compliance with transfection protocols.

4. Accountability

The Head of Cell Line Development is accountable for ensuring all transfection troubleshooting actions are documented, compliant, and scientifically justified.

5. Procedure

5.1 Identification of Transfection Failure

1. Determine failure based on key indicators:
   • Low or no expression of reporter or target protein (e.g., GFP, antibody)
   • Reduced cell viability post-transfection
   • Clump formation or detachment in adherent cultures
2. Record observations in Transfection Observation Log (Annexure-1).

5.2 Checklist for Troubleshooting

1. Perform stepwise evaluation of the following:
   • Cell Health: Check passage number, morphology, confluency, and doubling time. Avoid over-confluent or senescent cells.
   • Transfection Reagent: Verify expiry, lot number, and storage condition. Perform reagent-only control tests if needed.
   • Plasmid DNA: Confirm concentration and purity (A260/A280 ratio ~1.8–2.0). Run agarose gel for integrity.
   • Complex Formation: Ensure proper incubation time, ratio (e.g., 3:1 Lipofectamine:DNA), and mixing procedure.
   • Media Conditions: Avoid serum or antibiotics during transfection. Use fresh, warmed Opti-MEM or equivalent medium.
   • Incubation Conditions: Ensure temperature, CO₂, and humidity within optimal ranges.

5.3 Root Cause Analysis

1. Use the 5 Whys or Fishbone Diagram to identify root causes.
2. Document root cause analysis in Transfection RCA Form (Annexure-2).

5.4 Experimental Re-Design

1. Based on findings, adjust:
   • DNA quantity and reagent ratio
   • Cell seeding density
   • Incubation time post-transfection
2. Record optimization steps and outcomes in the Transfection Optimization Sheet (Annexure-3).

5.5 Documentation and Reporting

1. Submit completed annexures to QA.
2. If deviation is logged, follow SOP/QA/012/2025 for deviation closure.
3. Ensure trend data is analyzed quarterly for repeated failure patterns.

6. Abbreviations

• CLD: Cell Line Development
• QA: Quality Assurance
• RCA: Root Cause Analysis

7. Documents

1. Transfection Observation Log (Annexure-1)
2. Transfection RCA Form (Annexure-2)
3. Transfection Optimization Sheet (Annexure-3)

8. References

• ICH Q10 – Pharmaceutical Quality System
• WHO TRS 978 – Annex 3: Biotech Products
• Internal SOP/QA/012/2025 – Deviation Management

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 Observation Log

Date	Cell Line	Method	Viability	Expression Observed	Operator
01/05/2025	CHO-K1	Lipofectamine 3000	72%	None	Ajay Verma

Annexure-2: Transfection RCA Form

Transfection ID	Issue	Root Cause	Investigated By	Date
TXN-055-001	Low GFP signal	Degraded plasmid	Sunita Reddy	02/05/2025

Annexure-3: Transfection Optimization Sheet

Parameter	Original	Modified	Result	Operator
DNA (µg)	2	3	Improved	Vinay Pawar

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Added structured troubleshooting steps and optimization log	Operational effectiveness