Standard Operating Procedure for Oxygen Enrichment Protocols in Biosimilar Bioreactor Operations

Department	Biosimilars
SOP No.	SOP/BS/105/2025
Supersedes	SOP/BS/105/2022
Page No.	Page 1 of 11
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To define the protocol for controlled oxygen enrichment in bioreactor systems used for biosimilar production to maintain optimal dissolved oxygen (DO) levels required for healthy cell growth and viability.

2. Scope

This SOP applies to all pilot and production scale bioreactors in the upstream biosimilar manufacturing facility that utilize automated or manual oxygen enrichment systems.

3. Responsibilities

• Bioreactor Operator: Execute oxygen enrichment based on DO setpoints, monitor DO probes, and log enrichment events.
• Supervisor: Approve oxygen enrichment activation and ensure all parameters are within defined ranges.
• Engineering: Maintain oxygen supply lines and validate gas flow sensors.

4. Accountability

The Head of Manufacturing is accountable for ensuring safe and validated oxygen enrichment operations during upstream runs.

5. Procedure

5.1 Pre-Operation Checks

1. Ensure oxygen cylinders or centralized liquid oxygen tank is functional and connected securely.
2. Check for calibration of DO sensors and flowmeters before batch start.
3. Verify interlock between DO controller and mass flow controller (MFC) for oxygen line.

5.2 DO Control Setpoints

1. Set desired DO setpoint (e.g., 40% ± 5%) in the SCADA interface.
2. Allow default cascade control:
   • Air sparging (primary)
   • Agitation ramp-up
   • Oxygen enrichment (final tier)

5.3 Oxygen Enrichment Activation

1. If DO drops below threshold (<35%), oxygen enrichment is auto-triggered or may be manually started.
2. Manually enrich by opening the MFC or valve controlling oxygen line to maintain setpoint.
3. Do not exceed oxygen flow rate of 0.1 vvm unless approved by supervisor.

5.4 Monitoring During Enrichment

1. Continuously monitor:
   • DO trend graph
   • Cell viability
   • Foam generation
2. Log enrichment time, duration, oxygen flow rate in Annexure-1.

5.5 Safety and Alarms

1. Ensure all enrichment lines are fitted with non-return valves to avoid backflow.
2. Monitor for high DO alarms (>70%) and reduce oxygen immediately if triggered.
3. Report any leak or pressure anomalies to Engineering immediately.

5.6 Post-Operation

1. Close oxygen valves and purge lines after batch completion.
2. Clean oxygen sparger and verify MFC is within calibration (Annexure-2).

6. Abbreviations

• DO: Dissolved Oxygen
• MFC: Mass Flow Controller
• vvm: Volume of gas per volume of liquid per minute
• QA: Quality Assurance

7. Documents

1. Oxygen Enrichment Log – Annexure-1
2. Oxygen Line and MFC Check Record – Annexure-2

8. References

• ICH Q8 – Pharmaceutical Development
• FDA Guidance for Industry: Process Validation (2011)

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Oxygen Enrichment Log

Date	Batch No.	DO % Before	O₂ Flow Rate (vvm)	Start Time	End Time	Logged By
04/05/2025	BS-214	32%	0.05	10:20	11:00	Sunita Reddy

Annexure-2: Oxygen Line and MFC Check Record

Date	Equipment ID	Checked By	Calibration Due	Status
04/05/2025	MFC-203	Ajay Verma	31/07/2025	OK

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Included MFC safety checks and DO alarm thresholds	Process improvement