Standard Operating Procedure for Mycoplasma Testing in Biosimilar Cell Banks

Department	Biosimilars
SOP No.	SOP/BS/029/2025
Supersedes	SOP/BS/029/2022
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 procedure for performing validated mycoplasma testing of Master Cell Bank (MCB) and Working Cell Bank (WCB) samples to ensure biosafety and GMP compliance for biosimilar manufacturing.

2. Scope

This SOP applies to the Quality Control Microbiology department performing routine mycoplasma detection on cryopreserved cell banks using both culture-based and nucleic acid-based detection methods.

3. Responsibilities

• QC Analyst: Conducts mycoplasma testing using validated methods and maintains all records.
• QC Supervisor: Reviews data and ensures compliance with compendial methods.
• QA Officer: Ensures procedural adherence, documentation accuracy, and release control.

4. Accountability

The Head of Quality Control is accountable for the implementation of validated mycoplasma testing to safeguard product integrity and meet regulatory expectations.

5. Procedure

5.1 Sample Preparation

1. Retrieve a representative vial of MCB/WCB under aseptic conditions.
2. Thaw and dilute cells to achieve a concentration of 1×10⁶ cells/mL in sterile PBS or culture media.
3. Split sample into two aliquots for culture and PCR testing.

5.2 Culture Method (Direct Assay)

1. Inoculate 1 mL of cell suspension into:
   • Mycoplasma broth (e.g., SP4 or Hayflick medium)
   • Mycoplasma agar plates
2. Incubate broth at 35–37°C in a 5% CO₂ incubator for 14 days.
3. On Day 14, subculture from broth to agar for colony growth visualization.
4. Observe for typical “fried-egg” colonies under 100× magnification using inverted microscopy.

5.3 PCR-Based Detection

1. Extract DNA from 1 mL of sample using validated kits.
2. Use genus-specific primers for Mycoplasma spp.
3. Run PCR alongside positive (Mycoplasma fermentans DNA) and negative (nuclease-free water) controls.
4. Analyze products by agarose gel electrophoresis.

5.4 Interpretation and Reporting

1. No colonies on agar and no amplification on gel → Negative
2. Presence of colonies or PCR band → Positive (Contamination)
3. Record all findings in Mycoplasma Testing Log (Annexure-1).
4. Prepare Mycoplasma Test Report (Annexure-2).

5.5 Out of Specification (OOS) Handling

1. If contamination is confirmed:
   • Initiate OOS and investigation protocol.
   • Quarantine the respective cell bank batch.
   • Repeat testing using retained samples.

6. Abbreviations

• MCB: Master Cell Bank
• WCB: Working Cell Bank
• PCR: Polymerase Chain Reaction
• QC: Quality Control

7. Documents

1. Mycoplasma Testing Log (Annexure-1)
2. Mycoplasma Test Report (Annexure-2)

8. References

• European Pharmacopoeia 2.6.7 – Mycoplasmas
• USP <63> – Mycoplasma Tests
• WHO Guidelines on the Quality, Safety and Efficacy of Biotherapeutic Protein 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: Mycoplasma Testing Log

Date	Sample ID	Method	Result	Analyst	Remarks
03/05/2025	MCB-BS-029	Culture + PCR	Negative	Sunita Reddy	Compliant

Annexure-2: Mycoplasma Test Report

Sample ID	Bank Type	Test Method	Date Tested	Result	Approved By
MCB-BS-029	MCB	Culture + PCR	03/05/2025	Mycoplasma Not Detected

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Integrated PCR method with compendial testing requirements	Regulatory Update