Standard Operating Procedure for Adaptation of Cells to Serum-Free Media in Biosimilar Development

Department	Biosimilars
SOP No.	SOP/BS/016/2025
Supersedes	SOP/BS/016/2022
Page No.	Page 1 of 12
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To describe the step-by-step procedure for adapting mammalian cell lines used in biosimilar production from serum-containing media to serum-free media to support regulatory compliance, scalability, and safety.

2. Scope

This SOP applies to all R&D and process development personnel involved in upstream cell culture operations for biosimilars where serum-free media is required for clone expansion or bioreactor inoculation.

3. Responsibilities

• Cell Culture Scientist: Designs and monitors the adaptation process.
• Technician: Performs daily cell culture maintenance and media exchanges.
• QA Officer: Reviews adaptation logs and confirms compliance with procedural requirements.

4. Accountability

The Head of Upstream Process Development is accountable for ensuring successful adaptation of production clones to serum-free conditions and maintaining traceable documentation.

5. Procedure

5.1 Preparation and Planning

1. Select serum-free medium compatible with the specific cell line (e.g., CD-CHO, ProCHO5).
2. Prepare complete serum-containing medium and serum-free medium (SFM) in advance.
3. Seed cells at 3–5 × 10⁵ cells/mL in serum-containing medium in T-75 flasks.

5.2 Stepwise Adaptation Schedule

1. Begin with a 75:25 mixture of serum-containing medium to SFM.
2. Allow cells to reach ~80% confluency or 1 × 10⁶ cells/mL.
3. Subculture using the next ratio: 50:50, then 25:75, and finally 100% SFM in successive passages.
4. Monitor cell viability, morphology, and doubling time after each adaptation step.
5. If viability drops below 85%, repeat the previous step before advancing.

5.3 Maintenance in Serum-Free Medium

1. Once cells grow stably in 100% SFM for at least 3 passages, consider adaptation complete.
2. Expand adapted cells for productivity testing and bioreactor inoculum.
3. Document final adaptation status in the Adaptation Completion Log (Annexure-2).

5.4 Troubleshooting

1. For poor growth or clumping, supplement with minimal FBS (≤1%) for one cycle.
2. Increase passage interval or seeding density if doubling time is extended.
3. Ensure media components (e.g., L-glutamine) are not degraded during storage.

6. Abbreviations

• SFM: Serum-Free Medium
• FBS: Fetal Bovine Serum
• QA: Quality Assurance
• CHO: Chinese Hamster Ovary cells

7. Documents

1. Adaptation Tracking Log (Annexure-1)
2. Adaptation Completion Log (Annexure-2)

8. References

• ICH Q5D – Cell Substrate Guidelines
• WHO TRS 978 – Guidelines for Serum-Free Media
• CDSCO Guidelines on Animal-Origin-Free Cell Culture Systems

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Adaptation Tracking Log

Date	Passage No.	Medium Ratio	Viability (%)	Cell Density	Remarks
01/05/2025	P1	75:25	92	5.1 × 10⁵/mL	Normal growth
03/05/2025	P2	50:50	89	6.3 × 10⁵/mL	Slight lag

Annexure-2: Adaptation Completion Log

Clone ID	Media Used	Completion Date	Stable Passage No.	Adapted By	Remarks
CL-BS-016	CD-CHO	04/05/2025	P6	Sunita Reddy	Fully adapted

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Incorporated adaptation log format and troubleshooting section	Annual SOP review