Strategies for Efficient Analyses of Proteins: From Aggregates to Post-Translational Modifications

WEBINAR

This seminar will focus on strategies and techniques to maximize efficiency and resolution when using reversed phase chromatography (RPC) and size exclusion chromatography (SEC) in analyzing biomacromolecules. Aspects of method development and troubleshooting will be examined for both modes of chromatography.

At the conclusion of the seminar, the engaged attendee will learn:

• How to select and the importance of phase chemistry and particle morphology in analyzing biomolecules
• Best practices in choosing appropriate mobile phase systems for each mode of chromatography
• How to diagnose the source of problems when troubleshooting biomolecule separations

Analyzing biomacromolecules is a unique analytical challenge owing to a number of product-related impurities and the complexity of the analytes. This complexity is a result of the myriad number of modifications to these analytes in each sample including, but not limited to, phosphorylation, methylation, and glycosylation. Due to the heterogeneity of an individual analyte, in addition to a biomolecule’s tendency to interact with silica-based, reversed-phase stationary phases, the resulting chromatographic peak can exhibit extensive broadening and tailing. To further complicate matters, most large proteins do not ionize well using conventional electrospray ionization mass spectrometers. In addition, when a mixture of proteins is not well-resolved, chromatographically, prior to the mass spectrometer, further ion suppression can occur. At a higher level, sample heterogeneity also increases with aggregated and fragmented proteins. This is routinely determined by size exclusion chromatography, often lacking a deeper characterization of the impurities. Therefore, the need for highly efficient, robust, and reproducible analytical methods is not a suggestion, but a requirement, for accurate characterization of proteinaceous samples.