Compared to other charge-based separation techniques, iCIEF can usually separate protein charge variants in a fraction of the time required in contrast to a CIEF method on a conventional capillary electrophoresis instrument. Imaged capillary isoelectric focusing (iCIEF) has become an indispensable tool in therapeutic protein development and manufacturing because of its high analytical throughput, ease of use, fast method development, and excellent reproducibility. These changes affect target binding, biological activity, patient safety, and shelf life. Protein charge variants arise by deamidation, formation of N-terminal pyroglutamate, aggregation, isomerisation, sialylation, antibody fragmentation, and glycation of lysine residues. High-efficiency iCIEF charge variant peak separation and collection, followed by mass spectrometry (MS) characterisation or online iCIEF-MS with the National Institute of standards and Technology Humanized IgG1κ Monoclonal Antibody (NISTmAb) will be demonstrated.Ĭapillary isoelectric focusing, CIEF critical quality attribute, CQA imaged capillary isoelectric focusing, iCIEF post-translational modification, PTM multi attribute method, MAM ion exchange chromatography, IEC whole column image detection, WCID electro-osmotic flow, EOF.Īnalysing charge variants of therapeutic proteins is essential for the characterising and monitoring of critical quality attributes such as physicochemical and immunochemical properties, biological activity, and quantity during development and manufacturing. In this report, high-efficiency protein charge variant fractionation characterisation with a newly developed preparative iCIEF will be illustrated. Characterisation of these variants is required Various charge variants detected in the sample limit the application of iCIEF as a powerful tool in multi-attribute methods. Imaged capillary isoelectric focusing (iCIEF) is a high throughput, highly efficient separation technique in routine use by many biopharmaceutical companies for the determination of the charge heterogeneity of proteins.
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