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
- Verify readiness of seed culture (VCD ≥ 0.5 × 10⁶ cells/mL; viability ≥ 90%).
- Label shake flasks with media type, volume, batch ID, and inoculation date (Annexure-1).
- Ensure laminar air flow (LAF) cabinet is sterilized and validated before inoculation.
5.2 Flask Selection and Media Transfer
- Select sterile Erlenmeyer flasks of appropriate capacity (125 mL–1 L).
- Transfer pre-sterilized chemically defined media (CDM) to each flask under LAF conditions using sterile pipettes or transfer tubes.
- Fill flasks to 20–30% of total volume to allow adequate aeration.
5.3 Inoculation Procedure
- Mix the seed culture gently and aseptically withdraw the required volume.
- Inoculate each flask with calculated volume to achieve initial VCD of 0.2–0.5 × 10⁶ cells/mL.
- Close flask cap or cotton plug securely and place on sterile tray for transport.
5.4 Incubation and Monitoring
- Incubate shake flasks in a rotary shaker at 120–150 rpm, 37°C, and 5% CO₂.
- Record environmental parameters and flask placement (Annexure-2).
- Monitor VCD, viability, and morphology daily until the desired expansion is reached.
5.5 Contamination Control
- Submit air samples and flask surface swabs to microbiology as per SOP/BS/043/2025.
- 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
- Shake Flask Label Record – Annexure-1
- Incubation Log Sheet – Annexure-2
- 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 |