Standard Operating Procedure for Colony Screening and Expression Analysis in Biosimilar Cell Line Development

Department	Biosimilars
SOP No.	SOP/BS/012/2025
Supersedes	SOP/BS/012/2022
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 standardized method for colony screening and initial expression analysis in transfected cell lines to identify and select high-producing biosimilar clones.

2. Scope

This SOP applies to R&D personnel involved in screening colonies derived from stable or transient transfection experiments during biosimilar cell line development.

3. Responsibilities

• Research Scientist: Designs screening strategy and conducts expression assays.
• Lab Technician: Maintains colonies and performs ELISA/western blot sample prep.
• QA Personnel: Ensures documentation of screening records and result traceability.

4. Accountability

The Head of Cell Line Development is accountable for ensuring that colony screening procedures are followed as per regulatory and experimental standards.

5. Procedure

5.1 Colony Observation and Selection

1. Observe colonies in 96-well plates under an inverted microscope.
2. Score colonies based on morphology, growth rate, and adherence (if adherent cells).
3. Mark colonies with good morphology and transfer to 24-well plates for further expansion.

5.2 Expansion and Media Collection

1. Grow selected colonies in complete growth medium in 24-well plates for 3–5 days.
2. Harvest supernatant (if secreted protein) or collect cell lysate (if intracellular protein).
3. Store samples at -20°C until analyzed.

5.3 Expression Analysis

1. Perform preliminary expression analysis using:
   • ELISA – for quantification of secreted protein
   • SDS-PAGE – for molecular weight estimation
   • Western Blot – for identity confirmation (if needed)
2. Document results in the Expression Screening Log (Annexure-1).

5.4 Selection Criteria

1. Select top 10–20% colonies showing highest expression levels and acceptable growth morphology.
2. Expand into 6-well plates or T-25 flasks and maintain with consistent passage records.

5.5 Data Review and Record Keeping

1. Review data with QA and assign Clone ID to selected colonies.
2. Update all records including source plate, expression level, and passage status in Clone Screening Summary Sheet (Annexure-2).

6. Abbreviations

• SOP: Standard Operating Procedure
• ELISA: Enzyme-Linked Immunosorbent Assay
• SDS-PAGE: Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
• QA: Quality Assurance

7. Documents

1. Expression Screening Log (Annexure-1)
2. Clone Screening Summary Sheet (Annexure-2)

8. References

• ICH Q5D – Cell Substrates
• WHO Guidelines on Recombinant Protein Production
• CDSCO Technical Manual for 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: Expression Screening Log

Date	Plate ID	Well No.	Clone ID	Assay Type	Result	Selected
03/05/2025	EX-01	D8	CL-BS-012	ELISA	72 ng/mL	Yes

Annexure-2: Clone Screening Summary Sheet

Clone ID	Source Plate	Expression (ng/mL)	Assay Type	Selected For Expansion	Remarks
CL-BS-012	EX-01:D8	72	ELISA	Yes	Top 5%

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Added new selection criteria and expanded annexures	Annual review