Direct Analysis of Glycosylated Therapeutics

Glycosylation plays a crucial role in determining the pharmacological properties of biotherapeutics. The molecular heterogeneity of glycosylated biotherapeutics makes it difficult to provide thorough characterization of intact glycoproteins by mass spectrometry. Current methods for analyzing biotherapeutics only measure fragments of the compound or rely on partial digestion of the glycans to reduce sample complexity. These limitations pose important risks and challenges for biotechnology companies that need to report human-compatible and consistent glycosylation, critical for drug safety and efficacy.

We will present a new approach to glycoform fingerprinting that uses proton-transfer charge-reduction with gas-phase fractionation to analyze intact glycoproteins by mass spectrometry. The method provides a detailed landscape of the intact molecular weights present in biotherapeutic protein preparations in a single experiment and offers insights into glycoform composition when coupled with a suitable bioinformatic strategy. In addition, we highlight the application of charge detection native mass spectrometry (Direct Mass Technology mode) towards the elucidation of masses and aggregation of heterogeneous glycosylated biotherapeutics.