Retracted:An integrated protocol based on workflows of imaged capillary isoelectric focusing (icIEF) for in-depth protein heterogenous characterization: High-efficient fractionation and online mass spectrometry detection

Abstract

Herein we describe an integrated protocol to study the charge heterogeneity of therapeutic proteins. The protocol integrating two workflows, based on imaged capillary isoelectric focusing (icIEF) with fractionation and mass spectrometry (MS) online detection on the same platform, is validated, and can be used for protein charge variant characterization at intact MS and in-depth peptide mapping of collected fractions by high performance liquid chromatography (HPLC). The developed protocol was evaluated based on systematic methodology validation, which is demonstrated to be a robust platform that suits for the heterogeneity characterization of diverse therapeutic proteins with Standard Operating Procedures (SOP), outstanding reproducibility and excellent sensitivity. Especially, the constructed protocol can perform both functions of icIEF fractionation and MS online detection in a unique icIEF platform with high throughput, easy method development and less reagent consumption as comparing to traditional technologies, which “Green Chemistry Concept” helps biopharma industry address urgent challenges during the whole process of discovery, quality control and manufacture release.