Standard Operating Procedure for Perfusion Culture Technique in Bioreactor Operations for Biosimilars

Department	Biosimilars
SOP No.	SOP/BS/075/2025
Supersedes	SOP/BS/075/2022
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 procedure for implementing and managing perfusion culture techniques in bioreactor operations for biosimilar production, ensuring high-density cell growth and continuous product harvest under GMP compliance.

2. Scope

This SOP applies to upstream operations using perfusion culture in wave bioreactors or stirred-tank reactors for continuous production of biosimilars involving mammalian cells.

3. Responsibilities

• Upstream Operators: Execute perfusion protocol, monitor key parameters, and document data.
• Engineering Team: Ensure proper setup of cell retention systems and pump calibrations.
• QA Personnel: Verify SOP adherence, review logs, and approve deviation handling.

4. Accountability

The Bioprocess Supervisor is accountable for ensuring perfusion systems are validated and operated as per approved process parameters.

5. Procedure

5.1 Preparation and Equipment Setup

1. Ensure bioreactor is cleaned, sterilized, and pre-calibrated.
2. Install the appropriate cell retention system (e.g., ATF, TFF, spin filter).
3. Connect feed pumps and harvest lines with sterile connectors and tubing.
4. Check pump flow calibration and sensor integrity (Annexure-1).

5.2 Inoculation and Initial Batch Phase

1. Inoculate culture at 0.3–0.5 × 10⁶ cells/mL and allow for initial batch phase (24–48 hours).
2. Monitor cell density and metabolite levels every 8 hours.

5.3 Initiation of Perfusion

1. Start continuous feed when cell density reaches 5 × 10⁶ cells/mL.
2. Begin perfusion rate at 1–2 reactor volumes per day (VVD), as per process design.
3. Maintain steady-state cell density using controlled bleed or dilution rate.

5.4 Real-Time Monitoring

1. Track VCD, viability, glucose, lactate, pH, and DO in real time using integrated sensors.
2. Log all values every 4 hours in Annexure-2.
3. Adjust feed rate or retention rate to optimize productivity.

5.5 Harvest and Termination

1. Harvest product continuously or intermittently from the permeate stream.
2. Terminate culture when viability drops below 70% or product concentration peaks.
3. Disassemble system and clean per SOP/BS/098/2025.

6. Abbreviations

• VVD: Volume Volumes per Day
• ATF: Alternating Tangential Flow
• TFF: Tangential Flow Filtration
• VCD: Viable Cell Density

7. Documents

1. Perfusion System Setup Checklist – Annexure-1
2. Perfusion Monitoring Log – Annexure-2

8. References

• ICH Q8 – Pharmaceutical Development
• WHO TRS 1025 – Biopharmaceutical Process Control
• SOP/BS/098/2025 – Bioreactor Cleaning and Sanitization

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Perfusion System Setup Checklist

Item	Status	Checked By	Date
ATF Filter Connection	OK	Sunita Reddy	04/05/2025
Pump Calibration	OK	Sunita Reddy	04/05/2025

Annexure-2: Perfusion Monitoring Log

Date	Time	VCD (10^6 cells/mL)	Glucose (g/L)	Viability (%)	Feed Rate (L/day)	Remarks
04/05/2025	10:00	7.2	1.9	92	1.5	Stable

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Added ATF/TFF setup steps and updated feed rate guidelines	Process Optimization