Standard Operating Procedure for Biosafety Assessment of Cell Lines in Biosimilars

Department	Biosimilars
SOP No.	SOP/BS/036/2025
Supersedes	SOP/BS/036/2022
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 procedures for conducting comprehensive biosafety assessment of mammalian cell lines used in biosimilar manufacturing, ensuring compliance with global regulatory standards for sterility, mycoplasma, and viral contamination.

2. Scope

This SOP applies to the biosafety, quality control, and cell line development teams involved in establishing Master Cell Banks (MCB) and Working Cell Banks (WCB) for biosimilar production processes.

3. Responsibilities

• QC Microbiologist: Conducts sterility and mycoplasma testing.
• QC Virologist: Performs viral detection assays.
• Cell Line Development Scientist: Coordinates sample collection and characterization.
• QA Officer: Reviews biosafety data and validates test results.

4. Accountability

The Head of Quality Control is accountable for ensuring all biosafety assessments are performed, documented, and meet regulatory expectations prior to cell bank qualification or use in GMP production.

5. Procedure

5.1 Sample Preparation

1. Collect cell suspension (≥10⁷ cells/mL) from mid-log phase cultures under aseptic conditions.
2. Aliquot 5–10 mL each for sterility, mycoplasma, and virus testing.
3. Label with cell line ID, passage number, date, and analyst initials.

5.2 Sterility Testing

1. Perform as per USP <71> using fluid thioglycollate medium (FTM) and soybean casein digest medium (SCDM).
2. Incubate at 20–25°C (SCDM) and 30–35°C (FTM) for 14 days.
3. Observe for turbidity, sediment, or microbial growth. Record in Sterility Log (Annexure-1).

5.3 Mycoplasma Testing

1. Use culture-based and PCR-based mycoplasma detection.
2. Culture: Incubate on Mycoplasma Agar and Broth for 7–10 days and observe colonies under inverted microscope.
3. PCR: Extract DNA and use universal mycoplasma primers; electrophorese amplicons to confirm absence of signal.
4. Record results in Mycoplasma Log (Annexure-2).

5.4 Viral Testing

1. Perform the following:
   • In vitro assay using indicator cell lines (e.g., MRC-5, Vero)
   • In vivo testing in suckling mice, embryonated eggs (as required)
   • Electron microscopy for retrovirus-like particles
   • PCR/RT-PCR for specific adventitious viruses
2. Evaluate cytopathic effects (CPE), hemadsorption, and fluorescence signals.
3. Document in Viral Testing Log (Annexure-3).

5.5 Genetic Characterization

1. Perform STR (Short Tandem Repeat) profiling to authenticate cell line identity.
2. Compare STR profile against reference cell line database.
3. Confirm presence of target expression cassette by PCR or Southern blot.

5.6 Acceptance Criteria

1. No microbial growth observed during sterility tests.
2. No mycoplasma contamination detected in both culture and PCR tests.
3. No viral activity or CPE observed in in vitro/in vivo assays.
4. STR identity match ≥90% with reference standard.

5.7 Documentation

1. Compile all logs into a Biosafety Assessment Report (Annexure-4).
2. Submit report for QA review and approval prior to cell bank release.

6. Abbreviations

• STR: Short Tandem Repeat
• MCB: Master Cell Bank
• WCB: Working Cell Bank
• CPE: Cytopathic Effect

7. Documents

1. Sterility Log (Annexure-1)
2. Mycoplasma Detection Log (Annexure-2)
3. Viral Testing Log (Annexure-3)
4. Biosafety Assessment Report (Annexure-4)

8. References

• ICH Q5D – Cell Substrate Derivation and Characterization
• USP <71> – Sterility Tests
• WHO TRS 978 – Requirements for Biological Products

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Sterility Test Log

Date	Cell Line ID	FTM Result	SCDM Result	Tested By	Remarks
02/05/2025	CLD-036-A	No Growth	No Growth	Rajesh Kumar	Pass

Annexure-2: Mycoplasma Detection Log

Date	Cell Line	Culture Result	PCR Result	Remarks
02/05/2025	CLD-036-A	Negative	Negative	Pass

Annexure-3: Viral Testing Log

Date	Cell Line	Test Method	Result	Remarks
02/05/2025	CLD-036-A	In Vitro + EM	Negative	Pass

Annexure-4: Biosafety Assessment Report

Cell Line	Passage No.	Sterility	Mycoplasma	Viral	Genetic Identity	Conclusion
CLD-036-A	P9	Pass	Pass	Pass	Matched	Qualified

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Added STR profiling and PCR methodology	Regulatory enhancement