Standard Operating Procedure for Preparation of Seed Culture in Biosimilar Production
| Department | Biosimilars |
|---|---|
| SOP No. | SOP/BS/061/2025 |
| Supersedes | SOP/BS/061/2022 |
| Page No. | Page 1 of 10 |
| Issue Date | 04/05/2025 |
| Effective Date | 06/05/2025 |
| Review Date | 04/05/2026 |
1. Purpose
To define the standard procedure for preparation of seed culture from cryopreserved cell banks for initiation of biosimilar upstream fermentation processes in GMP-controlled environments.
2. Scope
This SOP applies to all upstream operations involved in seed culture generation using CHO, HEK293, or other mammalian cells for biosimilar production, from thawing to expansion in shake flasks.
3. Responsibilities
- Upstream Operators: Execute revival, expansion, and transfer operations as per protocol.
- QA: Verify documentation and compliance with GMP requirements.
- Process Lead: Approves seed culture batch for scale-up.
4. Accountability
The Upstream Process Head is accountable for seed culture readiness, ensuring its viability, purity, and compliance with biosimilar production standards.
5. Procedure
5.1 Preparation
- Ensure the seed culture room is cleaned, disinfected, and ready for operation as per SOP/BS/047/2025.
- Review MCB/WCB log to confirm the vial to be used.
- Label all required flasks and reagents.
5.2 Thawing of Cryopreserved Vial
- Retrieve cryovial from LN2 storage and record retrieval time in Annexure-1.
- Thaw vial quickly in a 37°C water bath for ~90 seconds with gentle agitation.
- Immediately transfer contents to 10 mL pre-warmed medium in a 15 mL sterile centrifuge tube.
- Centrifuge at 1000 rpm for 5 minutes to remove DMSO.
5.3 Inoculation into T-25 Flask
- Resuspend pellet in 5–10 mL of growth media (e.g., CD-CHO).
- Transfer into T-25 flask and incubate at 37°C, 5% CO2.
- Observe daily for cell attachment (for adherent lines) or density increase (suspension lines).
5.4 Scale-Up in Shake Flasks
- Once VCD ≥ 0.5 × 106 cells/mL with viability ≥ 90%, passage into 125 mL flask with 30 mL media.
- Expand sequentially to 250 mL and then 1 L flasks as per seed train strategy.
5.5 In-Process Monitoring
- Record VCD, viability, and pH daily in Annexure-2.
- Ensure no microbial contamination by daily microscopy and Gram staining (as per SOP/BS/043/2025).
5.6 Approval for Scale-Up
- Submit batch-wise performance data for QA review.
- Process Lead to authorize batch for bioreactor inoculation (Annexure-3).
6. Abbreviations
- VCD: Viable Cell Density
- MCB: Master Cell Bank
- WCB: Working Cell Bank
- DMSO: Dimethyl Sulfoxide
7. Documents
- Seed Culture Retrieval Log – Annexure-1
- Daily Cell Monitoring Sheet – Annexure-2
- Seed Batch Release Form – Annexure-3
8. References
- ICH Q5D – Cell Substrate Characterization
- WHO TRS 999 – Annex 2: Biotech Manufacturing Practices
- Internal Cell Bank Handling SOP (SOP/BS/025/2025)
9. SOP Version
Version: 2.0
10. Approval Section
| Prepared By | Checked By | Approved By | |
|---|---|---|---|
| Signature | |||
| Date | |||
| Name | |||
| Designation | |||
| Department |
11. Annexures
Annexure-1: Seed Culture Retrieval Log
| Date | Vial ID | Storage Location | Retrieved By | Time |
|---|---|---|---|---|
| 03/05/2025 | WCB-CHO-013 | Dewar-4 | Rajesh Kumar | 09:15 AM |
Annexure-2: Daily Cell Monitoring Sheet
| Date | VCD (×106/mL) | Viability (%) | pH | Microscopy Status |
|---|---|---|---|---|
| 03/05/2025 | 0.48 | 92 | 7.1 | No Contamination |
Annexure-3: Seed Batch Release Form
| Seed Batch ID | SD-CHO-105 |
|---|---|
| Prepared By | Sunita Reddy |
| Viability (%) | 94% |
| Approved By | |
| Release Date | 04/05/2025 |
Revision History:
| Revision Date | Revision No. | Revision Details | Reason for Revision | Approved By |
|---|---|---|---|---|
| 04/05/2025 | 2.0 | Updated thawing and monitoring instructions | Process Optimization |