A ‘Coomassie for Carbohydrates” - novel methodology for rapid detection and quantitation of glycosylated IgGs on an SDS-PAGE gel

Abstract

Abstract Glycosylation is a common post-translation modification present on majority of eukaryotic proteins. Therefore, it is dependent on the host cell line and affected by the cell culture and purification process. Additionally, glycosylation of therapeutic biologics is known to have a profound impact on binding and downstream effector functions. Hence, glycosylation is increasingly considered a critical quality attribute (CQA) of biopharmaceutical drugs and has been showed to affect the safety and efficacy of these molecules. Therefore, regulatory agencies have adopted a Quality by Design (QbD) approach for glycan monitoring at various steps with stringent controls to ensure efficacy and safety. This requires multiple, orthogonal fit-for-purpose tools to study the process. One key unmet need in downstream organizations of large pharmaceutical companies is a real-time, rapid glycan detection and quantification tool to guide purification/expression process. To meet this need, we have developed a selective, colorimetric assay which selectively stains sugar moieties (glycosylated IgGs) which appears as magenta bands on an SDS-PAGE resolved gel. The selectivity arises because the method leverages stereochemical differences of hydroxyl groups between amino acids and carbohydrates to selectively stain the primary alcohol in carbohydrates but not in amino acids. Specifically, our method selectively oxidizes the ‘cis-diol’ group in carbohydrates to aldehydes and stains the aldehydes on an SDS-PAGE gel by Schiff’s reaction. Our method is semi-quantitative and has comparable sensitivity to Coomassie with LoD of around 50ng. The method can detect glycosylated IgGs produced in multiple cell lines (including HEK and CHO), thereby enabling us to study batch-to-batch variability and can critically stain both N-linked and O-linked glycosylation. We also show that the assay can be used as a downstream purification guide for process analytics following ion-exchange and hydrophobic interaction chromatography.