Standard Operating Procedure for Gene Copy Number Determination in Biosimilar Cell Lines

Department	Biosimilars
SOP No.	SOP/BS/017/2025
Supersedes	SOP/BS/017/2022
Page No.	Page 1 of 13
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To establish a standardized method for determining the gene copy number of integrated biosimilar transgenes in cell lines using quantitative PCR (qPCR), aiding in clone selection and stability assessment.

2. Scope

This SOP applies to molecular biology and analytical development teams responsible for evaluating and validating biosimilar-producing clones through gene copy number quantification.

3. Responsibilities

• Molecular Biologist: Conducts DNA extraction, qPCR setup, and data analysis.
• Research Associate: Assists in sample preparation and maintains documentation.
• QA Officer: Verifies results and checks data integrity for regulatory compliance.

4. Accountability

The Head of Molecular Biology is accountable for ensuring accuracy and reproducibility of gene copy number data in biosimilar cell line evaluations.

5. Procedure

5.1 Genomic DNA Extraction

1. Harvest cells (≥1 × 10⁶) and wash with PBS.
2. Extract DNA using silica column-based kits or phenol-chloroform method.
3. Quantify and assess DNA purity using spectrophotometer (A260/A280 ratio 1.8–2.0).

5.2 Primer and Probe Design

1. Design primers targeting a region of the biosimilar transgene (e.g., Fc region for mAb genes).
2. Select a single-copy reference gene (e.g., β-actin or GAPDH).
3. Validate primer efficiency (90–110%) and specificity prior to use.

5.3 Real-Time PCR Setup

1. Prepare 20 µL reactions containing:
   • 10 µL SYBR Green or probe-based master mix
   • 1 µL each of forward and reverse primers (10 µM)
   • 1 µL genomic DNA (10–50 ng)
   • 7 µL nuclease-free water
2. Run samples in triplicates on a real-time PCR machine (e.g., ABI 7500).
3. Thermal cycling conditions:
   • Initial denaturation: 95°C for 10 min
   • 40 cycles: 95°C for 15 sec, 60°C for 60 sec

5.4 Data Analysis

1. Calculate ΔCt = Ct_transgene – Ct_reference.
2. Relative copy number = 2^-ΔΔCt compared to control clone or calibrator.
3. Include positive control (known copy number) and no template control (NTC).
4. Document results in Gene Copy Number Log (Annexure-1).

5.5 Acceptance Criteria

1. Coefficient of variation (CV) among replicates should be ≤5%.
2. Reference gene Ct range: 18–25.
3. Transgene Ct range: 18–30 depending on copy number.

6. Abbreviations

• qPCR: Quantitative Polymerase Chain Reaction
• DNA: Deoxyribonucleic Acid
• Ct: Cycle Threshold
• ΔCt: Difference in Ct values between target and reference genes

7. Documents

1. Gene Copy Number Log (Annexure-1)
2. qPCR Plate Map (Annexure-2)

8. References

• ICH Q6B – Test Procedures and Acceptance Criteria
• WHO TRS 1004 – Guidelines on Molecular Characterization
• MIQE Guidelines – Minimum Information for Publication of qPCR Experiments

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Gene Copy Number Log

Date	Clone ID	Ct (Target)	Ct (Reference)	ΔCt	Copy Number	Operator
03/05/2025	CL-BS-017	22.6	19.1	3.5	8	Sunita Reddy

Annexure-2: qPCR Plate Map

Well	Sample ID	Target Gene	Reference Gene	Replicate
A1	CL-BS-017	rhEGFR	GAPDH	1
A2	CL-BS-017	rhEGFR	GAPDH	2
A3	CL-BS-017	rhEGFR	GAPDH	3

Revision History:

Revision Date	Revision No.	Details	Reason for Revision	Approved By
04/05/2025	2.0	Updated primer efficiency criteria and added plate map annexure	Annual Review