Standard Operating Procedure for Selection of Stably Transfected Clones in Biosimilar R&D

Department	Biosimilars
SOP No.	SOP/BS/010/2025
Supersedes	SOP/BS/010/2022
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 process for selecting, isolating, and expanding stably transfected mammalian cell clones that integrate the gene of interest for long-term expression of biosimilar proteins.

2. Scope

This SOP applies to biosimilar R&D teams involved in generating stable cell lines for protein expression in systems such as CHO or HEK293 cells.

3. Responsibilities

• Research Scientist: Designs and executes selection strategy and performs screening assays.
• Lab Technician: Prepares selection medium and maintains cell cultures.
• QA Officer: Reviews clone data and ensures documentation integrity.

4. Accountability

The Head of Cell Line Development is accountable for ensuring stable clone selection complies with regulatory expectations and technical quality standards.

5. Procedure

5.1 Post-Transfection Recovery

1. Allow cells 24–48 hours of recovery in complete media post-transfection.
2. Monitor confluency and viability daily.

5.2 Antibiotic Selection

1. Prepare selection medium containing appropriate antibiotic (e.g., G418, Hygromycin, Puromycin) based on plasmid resistance marker.
2. Perform kill curve prior to experiment to determine minimum lethal dose.
3. Replace recovery medium with selection medium and incubate under standard conditions (e.g., 37°C, 5% CO₂).
4. Continue selection for 10–14 days until only resistant colonies remain.

5.3 Cloning and Isolation

1. Pick individual colonies manually using sterile pipette tips or cloning rings.
2. Transfer to 24-well plates containing selection media and incubate for 5–7 days.
3. Expand viable clones into larger wells or flasks gradually.

5.4 Screening for Expression

1. Harvest culture supernatant or cell lysate from expanded clones.
2. Evaluate expression levels using:
   • ELISA for secreted proteins
   • SDS-PAGE and Western blot for intracellular or membrane proteins
   • qPCR for gene copy number
3. Document findings in Clone Screening Log (Annexure-1).

5.5 Cryopreservation of Positive Clones

1. Select top-performing clones with stable expression.
2. Freeze vials using freezing media (10% DMSO in FBS or serum-free alternative).
3. Label with Clone ID, date, and passage number.
4. Store in -80°C initially, then transfer to liquid nitrogen for long-term storage.

5.6 Documentation and Records

1. Maintain complete records for each clone including origin, passage history, antibiotic used, expression data, and storage location.
2. Update Stable Clone Master Record (Annexure-2).

6. Abbreviations

• SOP: Standard Operating Procedure
• CHO: Chinese Hamster Ovary
• FBS: Fetal Bovine Serum
• ELISA: Enzyme-Linked Immunosorbent Assay
• DMSO: Dimethyl Sulfoxide

7. Documents

1. Clone Screening Log (Annexure-1)
2. Stable Clone Master Record (Annexure-2)

8. References

• ICH Q5D – Cell Substrates Used for Production
• WHO Guidelines for Cell Line Characterization
• CDSCO Handbook on Biologics

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 Screening Log

Date	Clone ID	Antibiotic	Expression Method	Result	Selected
03/05/2025	CL-CHO-25	G418	ELISA	High Expression	Yes

Annexure-2: Stable Clone Master Record

Clone ID	Plasmid	Passage No.	Storage Temp	Location	Remarks
CL-CHO-25	pCMV-rhEPO	18	LN2	CryoTank B2	Master Cell Bank

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Updated cryopreservation steps and added master record log	Annual SOP update