Standard Operating Procedure for Cryopreservation of Cell Banks in Biosimilars

Department	Biosimilars
SOP No.	SOP/BS/026/2025
Supersedes	SOP/BS/026/2022
Page No.	Page 1 of 12
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To outline the standardized procedure for cryopreservation of biosimilar-producing cell banks including Master Cell Bank (MCB) and Working Cell Bank (WCB), using GMP-compliant freezing and storage processes.

2. Scope

This SOP is applicable to the Biosimilars Cell Line Development team and associated QA/QC functions involved in preserving cell banks for long-term storage and production readiness.

3. Responsibilities

• CLD Technician: Prepares cell suspensions, prepares freezing media, and performs aliquoting.
• QA Officer: Verifies documentation, ensures compliance with aseptic and GMP requirements.
• Storage Custodian: Manages transfer and monitoring of cryovials in liquid nitrogen storage units.

4. Accountability

The Head of Cell Line Development is accountable for ensuring successful cryopreservation of cell banks following validated procedures and regulatory standards.

5. Procedure

5.1 Freezing Medium Preparation

1. Prepare freezing medium as per cell line requirements (e.g., 90% culture media + 10% DMSO).
2. Filter sterilize the freezing medium using 0.22 µm PVDF filters.
3. Label media containers with preparation date and expiry (use within 7 days).

5.2 Cell Suspension Preparation

1. Harvest cells at viability ≥95% during exponential growth phase.
2. Centrifuge at 300 × g for 5 min; discard supernatant and resuspend in cold freezing medium.
3. Adjust final concentration to 5 × 10⁶ cells/mL.

5.3 Cryovial Aliquoting

1. Label sterile cryovials with:
   • Cell line ID
   • Bank type (MCB/WCB)
   • Lot number
   • Date
   • Passage number
2. Dispense 1.0 mL of cell suspension per vial under laminar airflow.

5.4 Controlled Rate Freezing

1. Place vials in a freezing container or programmable freezer set at -1°C/min to -80°C.
2. Allow freezing for at least 12–18 hours at -80°C.
3. Do not disturb or move vials during this period to avoid thermal shock.

5.5 Transfer to Long-Term Storage

1. Transfer frozen vials to a vapor-phase liquid nitrogen tank (-150°C to -196°C).
2. Update inventory record with tank number, rack, and box position.
3. Document in Cryopreservation Log (Annexure-1).

5.6 Documentation and QC

1. Record:
   • Cell density and viability before freezing
   • Batch ID and date
   • Initial storage temperature
2. Send 2–3 vials for post-thaw viability testing.

6. Abbreviations

• DMSO: Dimethyl Sulfoxide
• MCB: Master Cell Bank
• WCB: Working Cell Bank
• CLD: Cell Line Development

7. Documents

1. Cryopreservation Log (Annexure-1)
2. Freezing Batch Record (Annexure-2)

8. References

• ICH Q5D – Guidelines on Cell Substrate Derivation and Banking
• WHO TRS 978 – GMP Guidelines for Cell Banks
• CDSCO Biosimilar Regulatory Guidelines

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Cryopreservation Log

Date	Cell Line ID	Type	Vials Prepared	Freezing Temp.	Storage Location	Operator
03/05/2025	CL-BS-026	MCB	125	-80°C	LN2-T1-R3-B2	Sunita Reddy

Annexure-2: Freezing Batch Record

Batch No.	Cell Density (×10⁶/mL)	Viability (%)	Media Used	DMSO %	Operator
FRZ-BS-026A	5.2	96	Serum-free CHO	10%	Rajesh Kumar

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Freezing log and storage control updated	GMP enhancement and audit alignment