Standard Operating Procedure for Thawing of Frozen Cell Stocks in Biosimilar Development
| Department | Biosimilars |
|---|---|
| SOP No. | SOP/BS/027/2025 |
| Supersedes | SOP/BS/027/2022 |
| Page No. | Page 1 of 11 |
| Issue Date | 04/05/2025 |
| Effective Date | 06/05/2025 |
| Review Date | 04/05/2026 |
1. Purpose
To define the procedure for safe, sterile, and effective thawing of frozen biosimilar-producing cell stocks including Master Cell Banks (MCB) and Working Cell Banks (WCB) for initiation of cell culture processes.
2. Scope
This SOP applies to the Cell Line Development and Upstream Process Development departments responsible for recovering cryopreserved cell lines for biosimilar manufacturing and R&D activities.
3. Responsibilities
- CLD/USP Operator: Carries out the thawing process and initiates post-thaw culture recovery.
- QA Representative: Ensures compliance with aseptic handling, documentation, and deviation recording.
4. Accountability
The Head of Biosimilars Cell Line Development is accountable for ensuring viability, sterility, and traceability during thawing operations of cell stocks.
5. Procedure
5.1 Pre-Thaw Checks
- Verify the vial to be thawed using:
- Cell line ID
- Bank type (MCB/WCB)
- Lot and vial number
- Confirm the storage location from the inventory log.
- Prepare:
- Water bath at 37°C
- Pre-warmed complete growth media (10–20 mL)
- Sterile conical tubes and pipettes
5.2 Thawing Procedure
- Remove the cryovial from liquid nitrogen storage using protective gloves and face shield.
- Immediately immerse the bottom of the vial in a 37°C water bath, holding the cap above water level.
- Swirl gently and monitor thawing. Once ice just melts (~1–2 minutes), immediately remove.
- Disinfect the vial exterior with 70% IPA before transferring to the biosafety cabinet.
5.3 Cell Transfer and Recovery
- Transfer thawed cell suspension into a 15 mL conical tube containing 10 mL of pre-warmed growth medium.
- Mix gently and centrifuge at 200 × g for 5 minutes to remove DMSO.
- Discard the supernatant and resuspend the pellet in fresh medium.
- Seed into appropriate culture vessel (T-25 or T-75 flask) and incubate at 36–37°C, 5% CO₂.
5.4 Post-Thaw Monitoring
- Check cell morphology and viability after 4–6 hours and on Day 1.
- Document recovery and cell count in Thawing Log (Annexure-1).
- Observe for signs of contamination or abnormal cell behavior.
6. Abbreviations
- MCB: Master Cell Bank
- WCB: Working Cell Bank
- DMSO: Dimethyl Sulfoxide
- IPA: Isopropyl Alcohol
7. Documents
- Thawing Log Sheet (Annexure-1)
8. References
- ICH Q5D – Cell Substrate Derivation and Characterization
- WHO Guidelines on Cell Banking and Handling
- CDSCO Biosimilar Regulatory Framework
9. SOP Version
Version: 2.0
10. Approval Section
| Prepared By | Checked By | Approved By | |
|---|---|---|---|
| Signature | |||
| Date | |||
| Name | |||
| Designation | |||
| Department |
11. Annexures
Annexure-1: Thawing Log Sheet
| Date | Vial ID | Cell Line | Bank Type | Recovery Viability (%) | Operator | Remarks |
|---|---|---|---|---|---|---|
| 03/05/2025 | WCB-BS-027-001 | CL-BS-027 | WCB | 93% | Sunita Reddy | Normal morphology |
Revision History:
| Revision Date | Revision No. | Revision Details | Reason for Revision | Approved By |
|---|---|---|---|---|
| 04/05/2025 | 2.0 | Added post-thaw monitoring requirements | Regulatory enhancement |