Abstract
Proteins are increasingly used as therapeutics. Their characterization is challenging due to their size and inherent heterogeneity notably caused by post-translational modifications, among which glycosylation is probably the most prominent. The glycosylation profile of therapeutic proteins must therefore be thoroughly analyzed. Here, we illustrate how the use of a combination of various cutting-edge LC or LC/MS(/MS) methods, and operating at different levels of analysis allows the comprehensive characterization of both the N- and O-glycosylations of therapeutic proteins without the need for other approaches (capillary electrophoresis, MALDI-TOF). This workflow does not call for the use of highly specialized/custom hardware and software nor an extensive knowledge of glycan analysis. Most notably, we present the point of view of a contract research organization, with the constraints associated to the work in a regulated environment (GxP). Two salient points of this work are i) the use of mixed-mode chromatography as a fast and straightforward mean of profiling N-glycans sialylation as well as an orthogonal method to separate N-glycans co-eluting in the HILIC mode; and ii) the use of widepore HILIC/MS to analyze challenging N/O-glycosylation profiles at both the peptide and subunit levels. A particular attention was given to the sample preparations in terms of duration, specificity, versatility, and robustness, as well as the ease of data processing.
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