Biosimilars: SOP for Clone Stability Assessment – V 2.0
Standard Operating Procedure for Clone Stability Assessment in Biosimilar Cell Line Development
| Department |
Biosimilars |
| SOP No. |
SOP/BS/023/2025 |
| Supersedes |
SOP/BS/023/2022 |
| 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 evaluating the genetic and phenotypic stability of biosimilar-producing clones across extended cell passages under laboratory conditions.
2. Scope
This SOP applies to Cell Line Development (CLD) and Analytical Development teams conducting stability studies on selected biosimilar clones before Master Cell Bank creation.
3. Responsibilities
- CLD Scientist: Maintains cell cultures across passages and prepares samples.
- Analytical Scientist: Performs expression testing, gene copy number analysis, and glycosylation profiling.
- QA Representative: Reviews test logs and verifies data consistency and documentation.
4. Accountability
The Head of CLD is accountable for ensuring that only genetically and phenotypically stable clones are advanced to cell banking and further development stages.
5. Procedure
5.1 Clone Expansion for Stability Study
- Thaw selected clone and culture in triplicate flasks.
- Maintain continuous passaging every 3–4 days for a total of 60 population doublings (or 30 passages).
- Document cell morphology, viability, and doubling time at each passage.
5.2 Sampling Strategy
- Collect cell samples at initial passage (P0), mid-point (P15), and final passage (P30).
- Preserve samples for:
- mRNA expression (RT-PCR)
- Protein expression (ELISA/Western blot)
- Gene copy number (qPCR)
- Glycan profiling (HPLC)
5.3 Expression Analysis
- Quantify biosimilar protein at each sampling point using validated method (e.g., ELISA).
- Compare final expression level with initial value. Acceptable drift: ±20%.
5.4 Gene Copy Number Analysis
- Extract genomic DNA and perform qPCR using transgene-specific primers.
- Use single-copy gene (e.g., GAPDH) for normalization.
- Document results in Clone Stability Log (Annexure-1).
5.5 Glycosylation Profile Monitoring
- Analyze glycan pattern via 2-AB labeled HPLC or lectin blotting.
- Ensure no major shift in sialylation or fucosylation profile.
5.6 Data Review and Acceptance Criteria
- Clone is considered stable if:
- Expression deviation ≤ ±20%
- Copy number deviation ≤ 1 copy
- Glycan profile remains within ±10% for major glycans
- Summarize findings in Stability Study Summary Report.
6. Abbreviations
- CLD: Cell Line Development
- qPCR: Quantitative Polymerase Chain Reaction
- HPLC: High Performance Liquid Chromatography
- 2-AB: 2-Aminobenzamide
7. Documents
- Clone Stability Log (Annexure-1)
- Expression Comparison Sheet (Annexure-2)
8. References
- ICH Q5B – Genetic Stability Testing Guidelines
- WHO TRS 999 – Biosimilar Clone Monitoring Standards
- CDSCO Guidance on Cell Line Characterization
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
| Clone ID |
Passage No. |
Expression (mg/L) |
Gene Copy No. |
Glycan Shift (%) |
Operator |
| CL-BS-023 |
P30 |
980 |
4.2 |
±6.3 |
Rajesh Kumar |
Annexure-2: Expression Comparison Sheet
| Passage |
Expression (mg/L) |
qPCR Copy Number |
Glycosylation Status |
Remark |
| P0 |
1000 |
4.5 |
Comparable |
Baseline |
| P15 |
970 |
4.3 |
No Shift |
Stable |
| P30 |
980 |
4.2 |
Minor Shift |
Stable |
Revision History:
| Revision Date |
Revision No. |
Revision Details |
Reason for Revision |
Approved By |
| 04/05/2025 |
2.0 |
Included glycosylation and qPCR-based assessment criteria |
Annual update and enhancement of stability protocol |
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