SOP Guide for Pharma

Biosimilars: SOP for Preparation of Seed Culture – V 2.0

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Biosimilars: SOP for Preparation of Seed Culture – V 2.0


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

See also  Biosimilars: SOP for Inoculation of Shake Flasks - V 2.0

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

  1. Ensure the seed culture room is cleaned, disinfected, and ready for operation as per SOP/BS/047/2025.
  2. Review MCB/WCB log to confirm the vial to be used.
  3. Label all required flasks and reagents.

5.2 Thawing of Cryopreserved Vial

  1. Retrieve cryovial from LN2 storage and record retrieval time in Annexure-1.
  2. Thaw vial quickly in a 37°C water bath for ~90 seconds with gentle agitation.
  3. Immediately transfer contents to 10 mL pre-warmed medium in a 15 mL sterile centrifuge tube.
  4. Centrifuge at 1000 rpm for 5 minutes to remove DMSO.

5.3 Inoculation into T-25 Flask

  1. Resuspend pellet in 5–10 mL of growth media (e.g., CD-CHO).
  2. Transfer into T-25 flask and incubate at 37°C, 5% CO2.
  3. Observe daily for cell attachment (for adherent lines) or density increase (suspension lines).
See also  Biosimilars: SOP for Dissolved Oxygen (DO) Control in Bioreactor Operations - V 2.0

5.4 Scale-Up in Shake Flasks

  1. Once VCD ≥ 0.5 × 106 cells/mL with viability ≥ 90%, passage into 125 mL flask with 30 mL media.
  2. Expand sequentially to 250 mL and then 1 L flasks as per seed train strategy.

5.5 In-Process Monitoring

  1. Record VCD, viability, and pH daily in Annexure-2.
  2. Ensure no microbial contamination by daily microscopy and Gram staining (as per SOP/BS/043/2025).

5.6 Approval for Scale-Up

  1. Submit batch-wise performance data for QA review.
  2. Process Lead to authorize batch for bioreactor inoculation (Annexure-3).

6. Abbreviations

7. Documents

  1. Seed Culture Retrieval Log – Annexure-1
  2. Daily Cell Monitoring Sheet – Annexure-2
  3. Seed Batch Release Form – Annexure-3

8. References

See also  Biosimilars: SOP for pH Monitoring and Adjustment in Bioreactor Operations - V 2.0

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
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