Standard Operating Procedure for Genetic Stability Testing in Biosimilar Cell Lines

Department	Biosimilars
SOP No.	SOP/BS/041/2025
Supersedes	SOP/BS/041/2022
Page No.	Page 1 of 12
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To define the standard procedure for assessing the genetic stability of biosimilar-producing cell lines through molecular and expression-level analyses to ensure consistent gene integration, copy number, and protein expression over multiple passages.

2. Scope

This SOP applies to all biosimilar projects involving stably transfected clones used for therapeutic protein production. It includes tests performed from the Master Cell Bank (MCB) to extended passages during process development.

3. Responsibilities

• Cell Line Development Team: Executes molecular assays and maintains records.
• QC Molecular Biology: Reviews and verifies data for regulatory submissions.
• QA Officer: Ensures documentation, compliance with protocols, and traceability.

4. Accountability

The Head of Process Development is accountable for the overall assurance that the genetic profile of the production cell line remains stable throughout its use.

5. Procedure

5.1 Cell Culture and Passage Plan

1. Recover cells from MCB vial and expand under standard culture conditions.
2. Pass cells to at least 60 generations, maintaining triplicate flasks per time point.
3. Harvest samples at predefined intervals (e.g., P0, P10, P30, P60) for analysis.

5.2 DNA Isolation

1. Isolate genomic DNA from 5×10⁶ cells using a validated DNA extraction kit.
2. Measure DNA concentration and purity using spectrophotometer (260/280 ratio).

5.3 PCR for Transgene Presence

1. Amplify gene of interest using specific primers.
2. Compare PCR amplicons across passage numbers using agarose gel electrophoresis.
3. Document gel images in Annexure-1.

5.4 Southern Blot Analysis

1. Digest genomic DNA with restriction enzymes.
2. Transfer DNA to membrane and probe with labeled gene-specific probe.
3. Assess consistency of banding patterns across all time points.

5.5 Quantitative PCR for Gene Copy Number

1. Perform qPCR using gene-specific and reference gene primers.
2. Calculate relative gene copy number using ΔΔCt method.
3. Acceptable variation: ±20% across passages.

5.6 Protein Expression and Productivity

1. Collect culture supernatants from each passage point.
2. Measure protein titer using ELISA or HPLC.
3. Compare expression data to initial baseline (MCB passage).

5.7 Criteria for Genetic Stability

1. No significant change in gene copy number.
2. Stable banding pattern in Southern blot.
3. Consistent protein expression across 60 generations.

5.8 Documentation

1. Record all raw data in the Genetic Stability Log (Annexure-2).
2. Maintain gel images, qPCR outputs, blot scans, and titer reports.

6. Abbreviations

• MCB: Master Cell Bank
• qPCR: Quantitative Polymerase Chain Reaction
• HPLC: High Performance Liquid Chromatography
• Ct: Cycle Threshold

7. Documents

1. Gel Image Log (Annexure-1)
2. Genetic Stability Log (Annexure-2)

8. References

• ICH Q5B – Genetic Stability of Recombinant DNA-Derived Products
• WHO Guidelines on Cell Substrates (TRS 978)
• FDA Guidance for Industry: Characterization of Cell Lines

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Gel Image Log

Date	Sample ID	Passage	Result	Image Reference
02/05/2025	CLD-GST-01	P10	Positive	Image-041-1

Annexure-2: Genetic Stability Log

Passage	Copy Number	Titer (g/L)	Band Pattern (Southern)	Comments
P0	6	1.20	Stable	Baseline
P30	6	1.18	Stable	No change
P60	5.9	1.15	Stable	Accepted

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Updated SOP with qPCR and HPLC-based productivity tracking	Process Optimization