PROTEIN AGGREGATION
ICH Q6B – Aggregates includes dimers and higher multiples of the desired product. These are generally resolved from the desired product and product related substances, and quantitated by appropriate analytical procedures (e.g., size exclusion chromatography, capillary electrophoresis)
CONSEQUENCES OF AGGREGATION
Aggregates can pose a risk to patient safety and product efficacy
REGULATORY EXPECTATIONS FOR SOLUBLE AGGREGATES
SIZE EXCLUSION CHROMATOGRAPHY - PRINCIPLE
Separation occurs based on size
SIZE EXCLUSION CHROMATOGRAPHY -INSTRUMENTATION
SEC MALS
- SEC-MALS combines multi-angle light scattering detector with size-exclusion chromatography.
- SEC-MALS determines molecular weight from 200 g/mol to 1 billion g/mol.
- It can also determine molecular size – the rms radius, or radius of gyration Rg – from 10 nm to 500 nm and beyond.
- By combining molar mass and size, it can also assess molecular conformation.
Applications:
- Absolute determination of molar mass
- Characterization of conjugated proteins like glycoproteins, pegylated proteins or surfactant-bound membrane proteins
- Determination of the stoichiometry of protein-protein and protein-nucleic acid complexes as well as native oligomers and aggregates
SEC MALS – Case Studies
Figure 1: Chromatograms of a) BSA and b) NIST mAb (Blue color chromatogram indicates UV response and red color chromatogram indicates MALS detector response)
SEC MALS ANALYSIS AT CBA
| Aggregate/Multimer analysis of proteins |
Sample requirement | Amount- 1-2 ml (0.5-1 mg/ml) Formulation buffer- 5 ml |
Deliverables | Size exclusion chromatogram of sample in UV and MALS mode, Percentage of monomer, dimer and other aggregates
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Information required from client |
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SERVICES OFFERED BY CBA
- Protein Intact Mass Analysis using HRMS
- Peptide Mapping using HRMS
- Post Translational Modifications Analysis using HRMS
- Host Cell Protein Impurity Identification using HRMS
- Host Cell Protein Impurity Profiling using SDS-PAGE
- Secondary Structure Analysis using far-UV CD Spectroscopy
- Tertiary Structure Analysis using near-UV CD Spectroscopy
- Tertiary Structure Analysis using Fluorescence Spectroscopy
- Higher Order Structure Analysis using Fluorescence Spectroscopy
- Secondary Structure Analysis using FTIR Spectroscopy
- Higher Order Structure Analysis using DSC
- Aggregate/Multimers Analysis using SEC-MALS
- Protein Protein Interaction Analysis using SPR
- Biosimilarity assessment Project
SERVICES OFFERED BY CBA
Dr. Smita Kale
PI- Center for BioPharma Analysis
Venture Center
100, NCL Innovation Park, Dr. Homi Bhabha Road, Pune – 08
Phone: 020-2586-5877 | Mobile: +91-9923147592
Email: smita@venturecenter.co.in
Mr. Yashodhan Shende
Business Development Executive
Venture Center
100, NCL Innovation Park, Dr. Homi Bhabha Road, Pune – 08
Phone: 020-2586-5877 | Mobile: +91-7755920096
Email: yashodhan@venturecenter.co.in
For more information, please contact:
Website: https://bioanalysis.in/