Standard Operating Procedure for Fed-Batch Cultivation Strategy in Bioreactor Operations for Biosimilars

Department	Biosimilars
SOP No.	SOP/BS/074/2025
Supersedes	SOP/BS/074/2022
Page No.	Page 1 of 13
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To define the process for executing fed-batch cultivation in bioreactor operations for biosimilar production, ensuring consistent cell growth, product yield, and regulatory compliance under GMP conditions.

2. Scope

This SOP applies to all fed-batch bioreactor campaigns used in mammalian cell culture for biosimilar manufacturing within the upstream processing department.

3. Responsibilities

• Bioprocess Operators: Execute feed protocols, monitor parameters, and document observations.
• Process Engineers: Design feed strategies and adjust based on culture performance.
• QA Personnel: Verify adherence to batch instructions and review feed records.

4. Accountability

The Head of Upstream Processing is accountable for ensuring fed-batch protocols are implemented as per validated procedures and deviations are managed appropriately.

5. Procedure

5.1 Pre-Cultivation Preparation

1. Ensure the bioreactor is pre-calibrated and sterile.
2. Prepare feed solutions (e.g., glucose, amino acids, lipids) as per formulation guidelines. Filter sterilize and store under refrigerated conditions.
3. Verify feed volumes and label feed vessels with batch number and composition (Annexure-1).

5.2 Inoculation and Batch Phase

1. Inoculate culture at 0.3–0.5 × 10⁶ cells/mL and monitor growth for 24–48 hours.
2. Maintain temperature, pH, DO, and agitation as per the master batch record.

5.3 Feed Initiation

1. Begin feeding once VCD reaches 1.0–1.5 × 10⁶ cells/mL or glucose falls below 2 g/L.
2. Start initial bolus feed of glucose (2%–4% final concentration) using sterile tubing or pump.

5.4 Continuous Feed Strategy

1. Implement feed as per predetermined schedule:
   • Fixed volume daily (e.g., 10 mL/L)
   • OR exponential feeding based on cell growth and metabolic needs
2. Monitor glucose, lactate, and ammonia levels every 12 hours.
3. Adjust feed concentration or timing based on nutrient consumption.

5.5 DO and pH Integration

1. Link DO feedback to agitation and oxygen cascade.
2. Ensure feed does not result in drastic pH drift; adjust with buffers or antifoam agents as necessary.

5.6 End of Cultivation

1. Stop feeding when VCD plateaus or viability drops below 80%.
2. Record total feed volume and culture parameters (Annexure-2).
3. Proceed to harvest as per SOP/BS/086/2025.

6. Abbreviations

• VCD: Viable Cell Density
• DO: Dissolved Oxygen
• GMP: Good Manufacturing Practice

7. Documents

1. Feed Solution Label – Annexure-1
2. Feed Monitoring Log – Annexure-2

8. References

• ICH Q8 – Pharmaceutical Development
• WHO TRS 999 – Guidelines for Biopharmaceutical Production
• SOP/BS/086/2025 – Harvest Process

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Feed Solution Label

Feed Type	Composition	Volume	Batch No.	Prepared By	Date
Feed A	Glucose 40%, Amino Acids	2L	FB-074-A	Rajesh Kumar	04/05/2025

Annexure-2: Feed Monitoring Log

Date	Time	Feed Volume (mL)	Glucose (g/L)	Lactate (mM)	Remarks
04/05/2025	12:00	100	2.1	1.8	Normal

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Expanded section on exponential feeding and integrated DO/pH control	Process Optimization