Standard Operating Procedure for Continuous Culture Protocol in Biosimilar Manufacturing

Department	Biosimilars
SOP No.	SOP/BS/143/2025
Supersedes	SOP/BS/143/2022
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 protocol for establishing and managing continuous culture (chemostat or perfusion) processes in biosimilar manufacturing, enabling consistent product quality and high productivity through steady-state control.

2. Scope

This SOP applies to upstream continuous culture systems implemented in process development and pilot-scale production for biosimilar products. It includes preparation, steady-state validation, monitoring, and deviation management.

3. Responsibilities

• Upstream Production Team: Execute and monitor continuous culture operations.
• Process Development: Design dilution rate and media strategy.
• QA: Review continuous run protocols, batch records, and deviation logs.

4. Accountability

The Head of Manufacturing is accountable for the validated implementation of continuous bioprocessing strategies under GMP-compliant systems.

5. Procedure

5.1 Preparation

1. Ensure reactor, sensors, and control systems are calibrated and qualified.
2. Prepare media reservoir with validated sterile media as per batch requirement.
3. Sterilize tubing sets, feed/harvest lines, and sample ports as per validated SIP procedure.

5.2 Inoculation and Initial Batch Phase

1. Inoculate bioreactor with seed culture and allow exponential batch growth until OD₆₀₀ or VCD reaches target for steady-state initiation.
2. Maintain optimal temperature, pH, DO, and agitation parameters.

5.3 Initiation of Continuous Feeding

1. Start fresh media feed using peristaltic or diaphragm pump.
2. Simultaneously initiate harvest at the same flow rate to maintain working volume.
3. Establish dilution rate (D) using formula:
   
   D = F/V
   
   Where F = feed rate (L/hr), V = working volume (L)

5.4 Steady-State Monitoring

1. Allow culture to reach steady-state (3–5 residence times).
2. Monitor and record:
   • OD₆₀₀ / VCD
   • Glucose, lactate, ammonia
   • Product titer (ELISA or HPLC)
   • Viability (≥ 90%)
3. Maintain data in Annexure-1: Continuous Run Logbook.

5.5 Sampling and Testing

1. Take sterile daily samples for in-process testing and trend charting.
2. Use Annexure-2: Sampling Record Sheet for documentation.

5.6 Deviation Handling

1. In case of:
   • Feed interruption
   • Pump failure
   • Contamination signs

   Stop process, isolate harvest, and notify QA immediately.

2. Document event in Annexure-3: Deviation Report Form.

6. Abbreviations

• OD: Optical Density
• VCD: Viable Cell Density
• DO: Dissolved Oxygen
• SIP: Steam In Place
• QA: Quality Assurance

7. Documents

1. Continuous Run Logbook – Annexure-1
2. Sampling Record Sheet – Annexure-2
3. Deviation Report Form – Annexure-3

8. References

• ICH Q8 – Pharmaceutical Development
• FDA PAT Guidance
• WHO TRS 1025 – Continuous Manufacturing of Biologics

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Continuous Run Logbook

Date	Time	VCD (x10⁶ cells/mL)	Viability (%)	Feed Rate (L/hr)	Harvest Rate (L/hr)
04/05/2025	10:00	5.6	93.2	1.5	1.5

Annexure-2: Sampling Record Sheet

Date	Sample ID	Analyte	Result	Analyst
04/05/2025	BS-CC-042	Glucose	2.3 g/L	Sunita Reddy

Annexure-3: Deviation Report Form

Date	Description	Action Taken	QA Notified	Status
04/05/2025	Feed pump stopped unexpectedly	Switched to backup pump	Yes	Open

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Included deviation log and annexures for sampling	Process Enhancement