Standard Operating Procedure for Oxygen Mass Transfer Rate Determination in Biosimilar Manufacturing

Department	Biosimilars
SOP No.	SOP/BS/141/2025
Supersedes	SOP/BS/141/2022
Page No.	Page 1 of 10
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To establish a validated procedure for determining the oxygen mass transfer rate (kLa) in biosimilar bioreactors to support process optimization, scale-up, and robust control of dissolved oxygen (DO) levels.

2. Scope

This SOP applies to all pilot-scale and production bioreactors used in upstream processing of biosimilars where DO is a critical process parameter. It includes both static and dynamic gassing-out methods for kLa determination.

3. Responsibilities

• Process Development: Design experiments and calculate kLa values for different bioreactor configurations.
• Engineering: Support setup, instrumentation, and parameter adjustments.
• QA: Review validation reports and ensure traceability of all records.

4. Accountability

The Head of Upstream Manufacturing is accountable for ensuring accurate kLa assessments are performed, documented, and incorporated into process development reports and scale-up protocols.

5. Procedure

5.1 Selection of Method

1. Use either:
   • Dynamic gassing-out method (preferred)
   • Sodium sulfite chemical method (alternative)
2. Select based on availability of DO sensors, reactor size, and automation capability.

5.2 Dynamic Gassing-Out Method

1. Fill bioreactor with WFI or buffer to working volume (no cells).
2. Deoxygenate media by sparging nitrogen until DO = 0%.
3. Switch to air sparging and record DO values at 10-second intervals until saturation.
4. Record airflow rate, agitation RPM, backpressure, and temperature.
5. Calculate kLa using:
   
   ln(C* – C) = -kLa × t
   
   Where C = DO concentration at time t, C* = saturated DO level.
6. Plot data on semi-logarithmic scale and derive slope for kLa.

5.3 Sodium Sulfite Method (if applicable)

1. Add 0.5 g/L sodium sulfite + 50 µM cobalt catalyst to WFI in the reactor.
2. Start air sparging and monitor oxygen uptake using DO probe or titration.
3. Calculate oxygen uptake rate and derive kLa based on oxygen consumption kinetics.

5.4 Documentation and Reporting

1. Record all raw readings, sensor calibration data, and calculations in Annexure-1: kLa Determination Log.
2. QA to review and archive validation data in equipment qualification file.

6. Abbreviations

• kLa: Oxygen mass transfer coefficient
• DO: Dissolved Oxygen
• WFI: Water for Injection
• QA: Quality Assurance

7. Documents

1. kLa Determination Log – Annexure-1
2. Sensor Calibration Certificate – Annexure-2

8. References

• ICH Q8 – Pharmaceutical Development
• FDA Guidance on Process Validation: General Principles and Practices
• USP <1046> – Cellular and Tissue-Based Products

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: kLa Determination Log

Date	Bioreactor ID	Method	RPM	Airflow	kLa (1/hr)	Performed By
04/05/2025	BR-500L	Dynamic	200	0.5 vvm	45	Ajay Verma

Annexure-2: Sensor Calibration Certificate

Sensor ID	Calibrated On	Calibrated By	Method	Certificate No.
DO-SN-038	03/05/2025	Sunita Reddy	Two-Point	CAL/025/2025

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Added sodium sulfite method as alternative option	Flexibility for validation