Standard Operating Procedure for Stable Cell Line Development in Biosimilar R&D

Department	Biosimilars
SOP No.	SOP/BS/015/2025
Supersedes	SOP/BS/015/2022
Page No.	Page 1 of 14
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To establish a standardized procedure for the development of stable mammalian cell lines expressing biosimilar therapeutic proteins. This includes transfection, clone selection, expansion, and characterization steps.

2. Scope

This SOP applies to biosimilar R&D personnel involved in generating and validating stable expression cell lines for preclinical and commercial biosimilar development.

3. Responsibilities

• Research Scientist: Designs and supervises the stable cell line generation workflow.
• Cell Culture Technician: Executes cell handling, selection, and clone expansion.
• QA Representative: Ensures adherence to procedural documentation and review of clone selection records.

4. Accountability

The Head of Cell Line Development is accountable for ensuring regulatory-compliant execution and traceability of stable cell line generation activities.

5. Procedure

5.1 Preparation for Transfection

1. Maintain parental cell lines (e.g., CHO, HEK293) in exponential growth phase.
2. Confirm cell viability (>90%) and absence of contamination.
3. Prepare transfection-grade plasmid DNA encoding the biosimilar gene of interest and selection marker.

5.2 Transfection

1. Transfect cells using electroporation or lipid-based reagent per protocol.
2. Plate transfected cells in T-75 or 6-well plates in antibiotic-free medium for 24–48 hours.

5.3 Antibiotic Selection

1. Determine kill curve for antibiotic (e.g., G418, Hygromycin) prior to application.
2. Add selection medium at predetermined lethal concentration and monitor for 10–14 days.
3. Change medium every 3–4 days.

5.4 Clone Isolation

1. Isolate single colonies using cloning rings or limiting dilution cloning.
2. Expand clones to 24-well plates, then to 6-well plates and T-25 flasks.
3. Maintain Clone Tracking Log (Annexure-1) throughout.

5.5 Expression Screening

1. Screen for expression using ELISA or Western blot.
2. Select top-performing clones with high and stable protein expression.
3. Document results in Expression Analysis Log (Annexure-2).

5.6 Clone Expansion and Characterization

1. Expand selected clones to generate enough biomass for further testing.
2. Confirm identity, gene copy number, productivity, and glycosylation profile.
3. Document characterization in Stable Clone Profile Sheet (Annexure-3).

5.7 Master Cell Bank (MCB) Generation

1. Freeze vials of confirmed stable clones under GMP-like conditions.
2. Label each vial with Clone ID, batch no., and date.
3. Store at -80°C and then transfer to liquid nitrogen storage.

6. Abbreviations

• CHO: Chinese Hamster Ovary
• MCB: Master Cell Bank
• ELISA: Enzyme-Linked Immunosorbent Assay
• QA: Quality Assurance

7. Documents

1. Clone Tracking Log (Annexure-1)
2. Expression Analysis Log (Annexure-2)
3. Stable Clone Profile Sheet (Annexure-3)

8. References

• ICH Q5D – Derivation and Characterization of Cell Substrates
• WHO Guidelines for Cell Line Development
• CDSCO Technical Guidelines on Cell Banks

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Clone Tracking Log

Date	Clone ID	Plate Type	Passage	Operator
02/05/2025	CL-BS-015	24-well	P2	Rajesh Kumar

Annexure-2: Expression Analysis Log

Clone ID	Method	Expression Level	Remarks	Date
CL-BS-015	ELISA	125 ng/mL	High Expressor	03/05/2025

Annexure-3: Stable Clone Profile Sheet

Clone ID	Gene Copy No.	Glycan Profile	MCB Created	MCB Location
CL-BS-015	8	Comparable to Reference	Yes	CryoTank A2

Revision History:

Revision Date	Revision No.	Details	Reason for Revision	Approved By
04/05/2025	2.0	Updated MCB generation steps and added Annexure-3	Annual Review