Standard Operating Procedure for Inoculation of Shake Flasks in Biosimilar Upstream Processing

Department	Biosimilars
SOP No.	SOP/BS/065/2025
Supersedes	SOP/BS/065/2022
Page No.	Page 1 of 11
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To outline the procedure for aseptic inoculation of shake flasks in biosimilar upstream processing to ensure optimal cell growth and contamination-free seed train scale-up.

2. Scope

This SOP applies to the inoculation of 125 mL, 250 mL, 500 mL, and 1L shake flasks used for scaling up seed cultures from cryopreserved or revived cell banks during biosimilar production.

3. Responsibilities

• Upstream Technician: Perform inoculation and record batch details.
• QA Personnel: Review logbooks, monitor environmental controls, and approve culture use.
• Microbiology Team: Conduct sterility checks on inoculated flasks and air samples.

4. Accountability

The Head of Upstream Process is accountable for ensuring the integrity of inoculated shake flasks and compliance with cGMP procedures for culture scale-up.

5. Procedure

5.1 Preparation Before Inoculation

1. Verify readiness of seed culture (VCD ≥ 0.5 × 10⁶ cells/mL; viability ≥ 90%).
2. Label shake flasks with media type, volume, batch ID, and inoculation date (Annexure-1).
3. Ensure laminar air flow (LAF) cabinet is sterilized and validated before inoculation.

5.2 Flask Selection and Media Transfer

1. Select sterile Erlenmeyer flasks of appropriate capacity (125 mL–1 L).
2. Transfer pre-sterilized chemically defined media (CDM) to each flask under LAF conditions using sterile pipettes or transfer tubes.
3. Fill flasks to 20–30% of total volume to allow adequate aeration.

5.3 Inoculation Procedure

1. Mix the seed culture gently and aseptically withdraw the required volume.
2. Inoculate each flask with calculated volume to achieve initial VCD of 0.2–0.5 × 10⁶ cells/mL.
3. Close flask cap or cotton plug securely and place on sterile tray for transport.

5.4 Incubation and Monitoring

1. Incubate shake flasks in a rotary shaker at 120–150 rpm, 37°C, and 5% CO₂.
2. Record environmental parameters and flask placement (Annexure-2).
3. Monitor VCD, viability, and morphology daily until the desired expansion is reached.

5.5 Contamination Control

1. Submit air samples and flask surface swabs to microbiology as per SOP/BS/043/2025.
2. Discard any contaminated flasks immediately as per SOP/BS/048/2025 and record in Deviation Log (Annexure-3).

6. Abbreviations

• VCD: Viable Cell Density
• CDM: Chemically Defined Media
• LAF: Laminar Air Flow
• RPM: Revolutions Per Minute

7. Documents

1. Shake Flask Label Record – Annexure-1
2. Incubation Log Sheet – Annexure-2
3. Deviation Log for Flask Contamination – Annexure-3

8. References

• ICH Q7 – GMP for Active Pharmaceutical Ingredients
• WHO TRS 999 – Guidelines on Biotherapeutic Products
• SOP/BS/043/2025 – Microbial Monitoring
• SOP/BS/048/2025 – Biohazardous Waste Disposal

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Shake Flask Label Record

Flask ID	Media Type	Volume (mL)	Inoculation Date	Batch ID
FLK-001	CD CHO	250	04/05/2025	SD-CHO-105

Annexure-2: Incubation Log Sheet

Date	Temperature (°C)	CO₂ (%)	RPM	Operator
04/05/2025	37.0	5.0	130	Ajay Verma

Annexure-3: Deviation Log for Flask Contamination

Date	Flask ID	Issue Observed	Action Taken	Reported By
04/05/2025	FLK-004	Turbidity with fungal growth	Discarded	Sunita Reddy

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Included incubation control points and contamination log format	Process Optimization