Standard Operating Procedure for Inoculation of Bioreactors in Biosimilar Manufacturing

Department	Biosimilars
SOP No.	SOP/BS/067/2025
Supersedes	SOP/BS/067/2022
Page No.	Page 1 of 12
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To establish a standardized procedure for inoculating bioreactors under aseptic conditions with qualified seed culture in biosimilar manufacturing, ensuring culture viability and contamination control.

2. Scope

This SOP applies to inoculation of stainless steel or single-use bioreactors ranging from 10L to 2000L, used for mammalian cell culture in upstream biosimilar production processes.

3. Responsibilities

• Upstream Process Operators: Conduct inoculation and record all parameters.
• Process Development Scientists: Approve seed culture quality and density for inoculation.
• QA Personnel: Verify environmental monitoring and documentation before and after inoculation.

4. Accountability

The Upstream Production Manager is accountable for ensuring that inoculation is performed under GMP conditions, using validated materials and equipment.

5. Procedure

5.1 Seed Culture Qualification

1. Confirm seed culture from spinner flask has VCD ≥ 1.0 × 10⁶ cells/mL and viability ≥ 90%.
2. Check for contamination indicators and record results in Seed Culture Log (Annexure-1).

5.2 Bioreactor Readiness

1. Verify that bioreactor sterilization (SIP or autoclave) is completed and filter integrity is confirmed.
2. Check media temperature (typically 36.5–37.5°C), pH (6.8–7.2), DO (setpoint 40–50%), and agitation speed.
3. Ensure vessel is labeled and configured as per batch record (Annexure-2).

5.3 Aseptic Inoculation

1. Perform inoculation inside Grade A/B environment or using sterile connection system (e.g., sterile weld or CPC connector).
2. Connect seed culture bag or transfer tubing aseptically to bioreactor inlet port.
3. Slowly transfer inoculum using gravity feed or peristaltic pump, avoiding shear or foaming.
4. Verify complete transfer and rinse transfer line with small volume of media if required.

5.4 Post-Inoculation Monitoring

1. Start agitation and monitor pH, DO, and temperature parameters continuously.
2. Take a sample 1 hour post-inoculation to check initial VCD, viability, and osmolality.
3. Perform microbial sampling of environment and personnel per SOP/BS/043/2025.

5.5 Documentation and Control

1. Record all inoculation data including transfer volume, time, method, and operator in Annexure-3.
2. Attach seed culture CoA and filter integrity reports to batch record.

6. Abbreviations

• VCD: Viable Cell Density
• SIP: Steam-In-Place
• DO: Dissolved Oxygen
• GMP: Good Manufacturing Practice

7. Documents

1. Seed Culture Qualification Log – Annexure-1
2. Bioreactor Readiness Checklist – Annexure-2
3. Inoculation Activity Log – Annexure-3

8. References

• ICH Q7 – Good Manufacturing Practice for Active Pharmaceutical Ingredients
• EU GMP Annex 1 – Manufacture of Sterile Medicinal Products
• SOP/BS/043/2025 – Microbiological Environmental Monitoring

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Seed Culture Qualification Log

Date	Flask ID	VCD (×10⁶/mL)	Viability (%)	Contamination Status
03/05/2025	SPF-002	1.1	91	Clear

Annexure-2: Bioreactor Readiness Checklist

Parameter	Target	Actual	Status
Temperature	37°C	36.9°C	OK
pH	7.0 ± 0.2	7.1	OK
DO	45%	44.8%	OK

Annexure-3: Inoculation Activity Log

Date	Bioreactor ID	Seed Volume (L)	Transfer Method	Operator
04/05/2025	BR-100L	5.0	Sterile Weld	Rajesh Kumar

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Added filter integrity and post-inoculation sampling requirements	Compliance Update