Biosimilars: SOP for Stability Study of Engineered Clones – V 2.0
Standard Operating Procedure for Stability Study of Engineered Clones in Biosimilars
| Department |
Biosimilars |
| SOP No. |
SOP/BS/035/2025 |
| Supersedes |
SOP/BS/035/2022 |
| Page No. |
Page 1 of 13 |
| Issue Date |
04/05/2025 |
| Effective Date |
06/05/2025 |
| Review Date |
04/05/2026 |
1. Purpose
To define the procedure for conducting stability studies on engineered biosimilar-producing clones to assess their genetic and expression stability during prolonged culture, storage, and production cycles.
2. Scope
This SOP applies to the cell line development and process development teams involved in monitoring recombinant clones over extended passages and post-cryopreservation conditions for biosimilar manufacturing.
3. Responsibilities
- Cell Line Development Scientist: Conducts serial passaging and evaluates expression stability.
- QC Molecular Analyst: Performs genetic confirmation through PCR and sequencing.
- QA Officer: Reviews data sets, trend analysis, and ensures protocol adherence.
4. Accountability
The Head of R&D is accountable for ensuring proper evaluation and documentation of clone stability prior to cell bank generation and regulatory filing.
5. Procedure
5.1 Stability Study Design
- Initiate study with 3 vials of selected clone from working cell bank (WCB).
- Passage cells serially for 30–60 generations under production culture conditions (shake flask or spinner flasks).
- Retain frozen aliquots at every 10th passage for interim analysis.
5.2 Expression Stability Testing
- Evaluate culture supernatant at P0, P10, P20, P30 using:
- ELISA for protein titer
- SDS-PAGE for band integrity
- HPLC for glycosylation or aggregation profile (optional)
- Compare results to initial expression data. Variability should be within ±20%.
5.3 Genetic Stability Testing
- Isolate genomic DNA at P0 and final passage (e.g., P30 or P60).
- Confirm transgene integrity using:
- PCR of expression cassette
- Southern blot or qPCR for copy number
- DNA sequencing (if applicable)
- Document no mutation, deletion, or rearrangement in critical regions.
5.4 Post-Thaw Stability
- Evaluate 3 freeze-thaw cycles from same clone batch for:
- Viability after thawing
- Recovery time to log-phase growth
- Expression titer compared to pre-freeze levels
5.5 Data Evaluation and Acceptance Criteria
- Clone is considered stable if:
- Expression remains within ±20% across passages
- No change in copy number or integration site
- No degradation in protein quality attributes
5.6 Documentation
- Record results in Clone Stability Log (Annexure-1).
- Summarize study outcome in Stability Report (Annexure-2) for QA approval.
6. Abbreviations
- PCR: Polymerase Chain Reaction
- WCB: Working Cell Bank
- ELISA: Enzyme-Linked Immunosorbent Assay
- SDS-PAGE: Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
7. Documents
- Clone Stability Log (Annexure-1)
- Stability Study Report (Annexure-2)
8. References
- ICH Q5D – Derivation and Characterization of Cell Substrates
- ICH Q5B – Expression Characterization of Recombinant Proteins
- WHO TRS 999 – GMP for Biologicals
9. SOP Version
Version: 2.0
10. Approval Section
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Prepared By |
Checked By |
Approved By |
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11. Annexures
Annexure-1: Clone Stability Log
| Passage No. |
Viability (%) |
Titer (g/L) |
Copy Number |
Remarks |
Scientist |
| P0 |
94.8 |
1.02 |
5.1 |
Baseline |
Sunita Reddy |
| P30 |
93.2 |
1.04 |
5.0 |
Stable |
Sunita Reddy |
Annexure-2: Stability Study Report
| Clone ID |
Tested Passages |
Expression Variance |
Genetic Result |
Conclusion |
Approved By |
| C-035-A3 |
P0–P30 |
±2.3% |
No Mutation |
Clone Stable |
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Revision History:
| Revision Date |
Revision No. |
Revision Details |
Reason for Revision |
Approved By |
| 04/05/2025 |
2.0 |
Included sequencing and freeze-thaw analysis |
Process validation update |
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