Standard Operating Procedure for pCO₂ and pO₂ Monitoring in Bioreactor Operations for Biosimilars

Department	Biosimilars
SOP No.	SOP/BS/083/2025
Supersedes	SOP/BS/083/2022
Page No.	Page 1 of 12
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To define the procedure for monitoring partial pressure of carbon dioxide (pCO₂) and oxygen (pO₂) during bioreactor operations for biosimilar production, using inline sensors and off-line validation to ensure cell culture health and productivity.

2. Scope

This SOP applies to all bioreactor systems in upstream processing of biosimilars that utilize real-time pCO₂ and pO₂ monitoring via probes or external analyzers.

3. Responsibilities

• Bioprocess Technicians: Monitor and log pCO₂ and pO₂ values routinely and escalate deviations.
• Instrumentation Engineer: Calibrate and maintain gas sensors and analyzers.
• QA Personnel: Review gas level trends and verify control actions.

4. Accountability

The Production Head is accountable for maintaining accurate pCO₂/pO₂ profiles during bioreactor culture and ensuring adherence to validated ranges.

5. Procedure

5.1 Sensor Setup and Calibration

1. Ensure pCO₂ and pO₂ sensors are installed, sterilized, and connected to the controller or SCADA system.
2. Calibrate sensors as per SOP/BS/082/2025 before each batch or after any probe replacement.
3. Verify baseline and slope using certified gas standards (5% CO₂, 21% O₂).

5.2 Online Monitoring

1. Start recording pCO₂ and pO₂ levels once inoculation is complete.
2. Typical acceptable values:
   • pO₂: Maintain 30%–70% of air saturation
   • pCO₂: Maintain below 100 mmHg (approx. 13.3 kPa)
3. Monitor trends continuously and log data every 2–4 hours in Annexure-1.
4. Adjust agitation, aeration, or CO₂ gassing based on deviations.

5.3 Off-line Validation (if required)

1. Collect 5 mL of culture fluid and transfer to blood gas analyzer under sterile conditions.
2. Compare off-line readings with inline sensor output to validate accuracy.
3. Document comparative data in Annexure-2.

5.4 Corrective Action

1. If pCO₂ exceeds limits:
   • Increase headspace aeration or use alkaline base to counter acidification.
2. If pO₂ drops:
   • Increase stirrer speed, sparger airflow, or add pure O₂ as needed.

5.5 Documentation

1. All readings must be recorded in Gas Monitoring Log (Annexure-1).
2. Any interventions or parameter changes must be authorized and recorded.

6. Abbreviations

• pO₂: Partial Pressure of Oxygen
• pCO₂: Partial Pressure of Carbon Dioxide
• SCADA: Supervisory Control and Data Acquisition
• QA: Quality Assurance

7. Documents

1. Gas Monitoring Log – Annexure-1
2. Off-line Validation Record – Annexure-2

8. References

• USP <1046> – Cell Viability and Growth
• ICH Q8 – Pharmaceutical Development

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Gas Monitoring Log

Date	Time	Batch No.	pO₂ (%)	pCO₂ (mmHg)	Action Taken	Operator
04/05/2025	14:00	BS-083-B01	54	87	Normal	Rajesh Kumar

Annexure-2: Off-line Validation Record

Date	Sample ID	Inline pO₂	Offline pO₂	Inline pCO₂	Offline pCO₂	Remarks
04/05/2025	GAS-058	52%	53%	89 mmHg	88 mmHg	Within limits

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Added off-line comparison section and CO₂ intervention strategy	Process Enhancement