Standard Operating Procedure for Creation of Master Cell Bank (MCB) in Biosimilars

Department	Biosimilars
SOP No.	SOP/BS/024/2025
Supersedes	SOP/BS/024/2022
Page No.	Page 1 of 14
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To outline the procedure for the preparation, qualification, documentation, and storage of Master Cell Banks (MCBs) derived from biosimilar-producing cell lines intended for GMP manufacturing.

2. Scope

This SOP applies to the Cell Line Development and Quality Control departments involved in the generation, testing, and archival of MCBs under GMP conditions.

3. Responsibilities

• CLD Scientist: Prepares and freezes MCB vials under controlled aseptic conditions.
• QC Microbiologist: Conducts sterility, mycoplasma, identity, and viability testing.
• QA Officer: Ensures all MCB-related activities are documented, reviewed, and approved as per GMP standards.

4. Accountability

The Head of Biosimilars is accountable for ensuring that the MCB is prepared, characterized, and stored in compliance with regulatory expectations and internal quality systems.

5. Procedure

5.1 Pre-Freezing Preparation

1. Use a genetically and phenotypically stable cell clone that has passed clone stability studies (refer SOP/BS/023/2025).
2. Thaw and expand the selected clone in a progressive scale-up from T-flasks → spinner flasks → production bioreactor (if applicable).
3. Monitor viable cell density (>90% viability) before harvest.

5.2 Cryopreservation Media

1. Prepare freezing medium: 90% basal media + 10% DMSO under sterile conditions.
2. Filter sterilize the medium using 0.22 µm filters.

5.3 MCB Aliquoting and Labeling

1. Resuspend cells at 5 × 10⁶ cells/mL in freezing media.
2. Dispense 1 mL into pre-labeled sterile cryovials (minimum 100 vials recommended).
3. Labels must include:
   • Cell Line ID
   • MCB Lot Number
   • Date of Freezing
   • Passage Number
   • Technician Initials

5.4 Controlled Freezing

1. Place vials into a programmable rate freezer: -1°C/min until -80°C.
2. Transfer to liquid nitrogen (-196°C) storage tank after 12–24 hours.

5.5 Quality Control Testing

1. Send representative vials for:
   • Cell viability
   • Sterility (as per USP <71>)
   • Mycoplasma (culture and PCR-based)
   • Cell line identity (STR profiling)
   • Adventitious virus testing (where required)
2. Record all results in MCB QC Log (Annexure-1).

5.6 Documentation and Archival

1. Maintain freezing records and storage inventory in MCB Inventory Log (Annexure-2).
2. Prepare MCB Certificate of Analysis upon completion of QC testing.

6. Abbreviations

• MCB: Master Cell Bank
• DMSO: Dimethyl Sulfoxide
• STR: Short Tandem Repeat
• QC: Quality Control

7. Documents

1. MCB QC Log (Annexure-1)
2. MCB Inventory Log (Annexure-2)

8. References

• ICH Q5D – Derivation and Characterization of Cell Substrates
• WHO Technical Report Series No. 978 – Cell Substrate Guidelines
• CDSCO Biosimilar Guidelines (India)

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: MCB QC Log

Test	Method	Result	Acceptance Criteria	Remarks
Viability	Trypan Blue	94%	≥ 85%	Pass
Sterility	USP <71>	Negative	No growth	Pass
Mycoplasma	PCR + Culture	Negative	Absent	Pass
STR Profile	Capillary Electrophoresis	Matched	100% match	Pass

Annexure-2: MCB Inventory Log

Vial No.	Cell Line ID	MCB Lot No.	Freezing Date	Location (Tank/Rack/Box)	Operator
MCB001	CL-BS-024	MCB-BS-24A	03/05/2025	T1/R2/B4	Sunita Reddy

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Updated storage practices and included STR and virus testing	Regulatory update and audit recommendation