Standard Operating Procedure for Cell Line Authentication in Biosimilars

Department	Biosimilars
SOP No.	SOP/BS/030/2025
Supersedes	SOP/BS/030/2022
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 validated procedure for cell line authentication used in biosimilar production using genetic, morphological, and cytogenetic methods to confirm cell identity and prevent cross-contamination.

2. Scope

This SOP applies to all cell lines used in biosimilar R&D, MCB/WCB generation, and manufacturing, and covers STR profiling, species-specific PCR, and optional karyotyping and isoenzyme analysis.

3. Responsibilities

• QC Molecular Analyst: Conducts STR, PCR, and other identity assays.
• QA Officer: Verifies authenticity results and approves authentication records.
• CLD Head: Ensures authentication is completed before use in production or submission.

4. Accountability

The Head of Quality Control is accountable for ensuring accurate identity testing of production cell lines and implementation of corrective actions in case of misidentification.

5. Procedure

5.1 Sample Collection

1. Collect 3–5 × 10⁶ cells from the test culture vial.
2. Pellet cells and store at -80°C or proceed directly to DNA extraction.
3. Record details in the Authentication Sample Log (Annexure-1).

5.2 STR Profiling (Human Cell Lines)

1. Extract genomic DNA using silica column-based kits or magnetic beads.
2. Amplify loci such as D5S818, D13S317, D7S820, TH01, vWA, CSF1PO using multiplex STR kits.
3. Run samples on capillary electrophoresis and analyze alleles using fragment analysis software.
4. Compare the STR profile with reference database or ATCC cell bank profile.

5.3 Species-Specific PCR (Non-Human Cell Lines)

1. Use validated primers for species ID: CHO, HEK293, Vero, BHK, etc.
2. Perform PCR under standardized conditions with positive/negative controls.
3. Visualize products by gel electrophoresis.

5.4 Karyotyping (Optional)

1. Arrest dividing cells with Colcemid.
2. Prepare chromosome spreads and stain with Giemsa.
3. Assess chromosomal number and structure under microscope (optional for regulatory submission).

5.5 Result Interpretation

1. Identity confirmed when:
   • STR matches reference profile (≥ 80% match).
   • PCR result matches expected species band size.
   • No contamination bands or abnormal profiles are observed.
2. Document results in Authentication Report (Annexure-2).

5.6 Frequency and Retesting

1. Perform authentication:
   • Before MCB creation
   • After major scale-up or genetic modification
   • Every 6–12 months during ongoing use

6. Abbreviations

• STR: Short Tandem Repeat
• PCR: Polymerase Chain Reaction
• MCB: Master Cell Bank
• WCB: Working Cell Bank

7. Documents

1. Authentication Sample Log (Annexure-1)
2. Cell Line Authentication Report (Annexure-2)

8. References

• ICH Q5D – Cell Substrates
• WHO TRS 978 – GMP for Cell Banks
• ATCC Standards – Cell Line Authentication

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Authentication Sample Log

Date	Sample ID	Cell Line	Method	Analyst	Remarks
03/05/2025	CL-BS-030	CHO-S	Species PCR	Rajesh Kumar	Valid Profile

Annexure-2: Cell Line Authentication Report

Sample ID	Cell Line	Authentication Method	Date Tested	Result	Approved By
CL-BS-030	CHO-S	Species PCR	03/05/2025	CHO Identity Confirmed

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Updated STR loci list and added karyotyping step	Expanded regulatory requirement