title: Microchip electrophoresis-SDS methods with high-resolution and silver stain sensitivity for quality screening and quantitation of protein products.
abstract: Two microchip electrophoresis (ME)-SDS methods have been developed for high throughput quantitation and quality screening of protein products. Both methods utilize a commercial microchip instrument to separate dodecyl sulfate-coated proteins within 1 min. In the high-resolution ME-SDS method, improved separation selectivity is achieved using a mixture of sieving polymers. Proteins of similar sizes, such as different fragment antigen-binding (Fab) assemblies can be readily resolved and individually quantified. A high-sensitivity ME-SDS method was also developed with sensitivity comparable to that of SDS-PAGE with silver staining. In this method, protein molecules are derivatized with a fluorescence reagent prior to analysis. LIF detection of the covalently attached fluorophore enables accurate quantitation of low-expressing proteins and detection of minor species at 0.04% level (1 ng/mL loading concentration). Both the high-resolution and the high-sensitivity ME-SDS methods can be applied to crude fermentation samples. The utilities of these methods in process development and formulation stability study are presented.
authors.affiliation: Integrated Biologics Profiling, Novartis Pharmaceuticals, Cambridge, MA, USA.
authors.forename: Hongling
authors.initials: H
authors.lastname: Han
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Chen
mesh_terms: D046209:Electrophoresis, Microchip / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005285:Fermentation; D005456:Fluorescent Dyes / Q000737:chemistry; D057166:High-Throughput Screening Assays / Q000379:methods; D057230:Limit of Detection; D011506:Proteins / Q000032:analysis*; D016622:Silver Staining
chemical_list: D005456:Fluorescent Dyes; D011506:Proteins