Standard Operating Procedure for Establishing Clone Performance Metrics in Biosimilar Cell Line Development

Department	Biosimilars
SOP No.	SOP/BS/059/2025
Supersedes	SOP/BS/059/2022
Page No.	Page 1 of 10
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To outline the methodology for defining, measuring, analyzing, and documenting clone performance metrics during the Cell Line Development (CLD) stage of biosimilar manufacturing.

2. Scope

This SOP is applicable to all CHO, HEK293, or other mammalian cell clones screened and evaluated for stable expression of recombinant biosimilar proteins. The process includes productivity, growth rate, viability, expression stability, and product quality attributes.

3. Responsibilities

• CLD Scientists: Perform experiments, collect data, and generate clone metric reports.
• Bioanalytics Team: Analyze supernatant samples for titer and product quality.
• QA: Verify metric consistency with regulatory requirements and ensure data integrity.

4. Accountability

The Head of CLD is accountable for ensuring that clone performance metrics are established using scientifically valid methods and are reproducible under development and manufacturing conditions.

5. Procedure

5.1 Identification of Key Performance Indicators (KPIs)

1. Define the following metrics for each clone:
   • Specific productivity (qP, pg/cell/day)
   • Volumetric productivity (mg/L)
   • Viable Cell Density (VCD)
   • Doubling Time
   • Viability (%)
   • Stability Index (expression at passage N vs. N+10)

5.2 Clone Screening Strategy

1. Conduct preliminary screening using 24-well or 96-well plates.
2. Use ELISA or HPLC to determine expression levels of recombinant protein.
3. Select top 10% clones based on volumetric productivity and viability for further assessment.

5.3 Experimental Setup for Metric Evaluation

1. Seed selected clones in triplicates in 125 mL shake flasks at standard seeding density (e.g., 0.3 x 10⁶ cells/mL).
2. Monitor and record the following for each clone:
   • Daily cell count and viability
   • Metabolites (glucose, lactate)
   • Supernatant harvest for titer estimation (Day 5–7)
3. Calculate productivity per cell using the formula:
   
   qP = Titer (pg) / (VCD x culture duration)

5.4 Expression Stability Assessment

1. Subculture clones up to 60–80 generations (e.g., 20 passages).
2. Evaluate expression at every 5^th passage to calculate the Stability Index.
3. Acceptable clones must retain ≥90% expression levels compared to passage 5.

5.5 Data Review and Documentation

1. Document all metric data in Clone Performance Evaluation Sheet (Annexure-1).
2. Compile summary in Clone Metric Summary Report (Annexure-2) for project review board.
3. QA to audit records for traceability and reproducibility.

6. Abbreviations

• CLD: Cell Line Development
• qP: Specific Productivity
• VCD: Viable Cell Density
• QA: Quality Assurance

7. Documents

1. Clone Performance Evaluation Sheet – Annexure-1
2. Clone Metric Summary Report – Annexure-2

8. References

• ICH Q5D – Cell Substrate Characterization
• WHO TRS 1004 – Annex 3: Biotech Guidelines
• FDA Guidance on Biosimilar Product Development

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 Performance Evaluation Sheet

Clone ID	qP (pg/cell/day)	VCD (x106/mL)	Viability (%)	Doubling Time (hr)	Titer (mg/L)
CL-CHO-105	24.5	2.1	97%	22	132

Annexure-2: Clone Metric Summary Report

Clone ID	Expression Stability (%)	Final Rank	Recommended for MCB?
CL-CHO-105	94%	1	Yes

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Added stability metrics and updated evaluation forms	Periodic Review