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 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.
- 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|
|Information required from client||