SOP Guide for Pharma

Biosimilars: SOP for Selection of Stably Transfected Clones – V 2.0

Auditor


Biosimilars: SOP for Selection of Stably Transfected Clones – V 2.0


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

See also  Biosimilars: SOP for Colony Screening and Expression Analysis - V 2.0

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.
See also  Biosimilars: SOP for Adaptation of Cells to Serum-Free Media - V 2.0

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

See also  Biosimilars: SOP for Plasmid Amplification and Purification - V 2.0

7. Documents

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

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: 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
Exit mobile version