ICH Q6B recommends diligent Critical Quality Attributes characterization connected to physicochemical characterization, binding and biological activity, quantity, and purity. Physicochemical characterization program will generally include composition determination, physical properties, and structure of the therapeutic protein.
Primary structure:
- Amino Acid Sequencing (Edman degradation)
- Peptide mapping / Intact mass (LC MS/MS)
- Disulphide bridge mapping (LC MS/MS)
- Amino acid composition according (Pharmacopeia method)
Higher-order structure:
- Circular dichroism
- Fourier transform infrared (FTIR) spectroscopy
- NMR (1D & 2D) 400 MHz
- Differential Scanning Calorimetry (DSC) Analysis
- HPLC SEC
- PTMs identification and site localization
Extinction coefficient:
- UV-VIS
Isoform Pattern/Electrophoretic & LC Patterns:
- CE and cIEF
- SDS PAGE (reducing and non-reducing)
- HPLC SEC
- HPLC HIC
- RP HPLC