Standard Operating Procedure for Glycosylation Analysis in Biosimilar Clone Selection
| Department | Biosimilars |
|---|---|
| SOP No. | SOP/BS/021/2025 |
| Supersedes | SOP/BS/021/2022 |
| Page No. | Page 1 of 15 |
| Issue Date | 04/05/2025 |
| Effective Date | 06/05/2025 |
| Review Date | 04/05/2026 |
1. Purpose
To define the procedure for analyzing glycosylation profiles of expressed biosimilar proteins in mammalian cell clones, facilitating clone selection based on critical quality attributes (CQAs).
2. Scope
This SOP applies to Analytical Development, Cell Line Development, and Quality Control teams involved in assessing glycan structures during biosimilar clone screening.
3. Responsibilities
- Analytical Scientist: Executes glycan release, labeling, and chromatography or mass spec analysis.
- CLD Scientist: Provides purified protein samples for analysis.
- QA Representative: Reviews and verifies analysis logs and compliance to regulatory expectations.
4. Accountability
The Head of Analytical Development is accountable for ensuring accurate glycan profiling and interpretation to guide clone selection in biosimilar programs.
5. Procedure
5.1 Sample Preparation
- Collect 0.5–1.0 mg purified biosimilar protein from each clone.
- Buffer-exchange to PBS if required using desalting columns.
- Record sample details in Glycosylation Sample Log (Annexure-1).
5.2 N-linked Glycan Release
- Denature protein at 95°C for 10 minutes in presence of SDS and DTT.
- Add NP-40 and PNGase F enzyme; incubate at 37°C for 18 hours.
5.3 Glycan Labeling
- Dry released glycans using vacuum concentrator.
- Label with 2-AB (2-aminobenzamide) or procainamide dye in sodium cyanoborohydride solution.
- Incubate at 65°C for 2 hours in the dark.
5.4 HPLC/UPLC Analysis
- Inject labeled glycans onto amide column (e.g., BEH Glycan) connected to HPLC/UPLC system.
- Run with gradient of acetonitrile and ammonium formate buffer.
- Acquire fluorescence signal and calculate relative peak areas.
5.5 Optional Mass Spectrometry
- Desalt labeled glycans using HILIC cartridges.
- Analyze using LC-MS or MALDI-TOF to determine glycan composition.
5.6 Lectin Blotting (Optional)
- Run protein sample on SDS-PAGE and transfer to membrane.
- Block membrane and incubate with biotinylated lectins (e.g., SNA, ConA).
- Detect with streptavidin-HRP and chemiluminescent substrate.
5.7 Data Interpretation and Reporting
- Assign glycan peaks using reference ladder or glycan database.
- Calculate % abundance of high mannose, complex, sialylated, and fucosylated glycans.
- Compare profiles with innovator molecule specifications.
- Record results in Glycan Profile Log (Annexure-2).
6. Abbreviations
- HPLC: High Performance Liquid Chromatography
- PNGase F: Peptide:N-Glycosidase F
- 2-AB: 2-Aminobenzamide
- SNA: Sambucus Nigra Agglutinin
7. Documents
- Glycosylation Sample Log (Annexure-1)
- Glycan Profile Log (Annexure-2)
8. References
- ICH Q6B – Test Procedures and Acceptance Criteria for Biotech Products
- WHO TRS 1004 – Glycosylation Evaluation Guidelines
- CDSCO Biosimilar Quality Guidelines (India)
9. SOP Version
Version: 2.0
10. Approval Section
| Prepared By | Checked By | Approved By | |
|---|---|---|---|
| Signature | |||
| Date | |||
| Name | |||
| Designation | |||
| Department |
11. Annexures
Annexure-1: Glycosylation Sample Log
| Date | Clone ID | Sample Volume (µL) | Protein Conc. (mg/mL) | Operator |
|---|---|---|---|---|
| 03/05/2025 | CL-BS-021 | 500 | 2.0 | Sunita Reddy |
Annexure-2: Glycan Profile Log
| Date | Clone ID | High Mannose (%) | Fucosylated (%) | Sialylated (%) | Total Glycan Diversity | Remarks |
|---|---|---|---|---|---|---|
| 04/05/2025 | CL-BS-021 | 12.5 | 82.1 | 5.4 | 8 major peaks | Acceptable match with reference |
Revision History:
| Revision Date | Revision No. | Revision Details | Reason for Revision | Approved By |
|---|---|---|---|---|
| 04/05/2025 | 2.0 | Included LC-MS and lectin blot as optional methods | Annual SOP review and method expansion |