3rd International Top-Down Proteomics Symposium

Increasing sensitivity and reducing carryover for IgG glycoform characterization with monolithic hydrophilic interaction liquid chromatography-mass spectrometry

Not scheduled

20m

VMP 6 / Philturm (Lecture Hall D)

Poster Poster Session 1

Speaker

Thomas Holmark (University of Amsterdam)

Description

Immunoglobulins are a class of proteins that are crucial for the immune response to fight diseases. They contain many different post-translational modifications, and in particular, changes in the glycosylation patterns of immunoglobulins subclass-G (IgGs) can be linked to disease progression. In our project we aim to develop capillary-based LC-MS methods to characterize intact IgG secreted by in-vitro cultivated B-cells aiming to profile the changes in IgG glycosylation profile as response to the exposure to different stimuli.

Traditional RPLC-MS based methods focusing on intact analysis fail to capture the full glycoform distribution as isomeric species and low abundance glycoforms cannot be characterized due to co-elution. HILIC separations represent a good alternative to separate the different glycans on intact IgGs and allow for a more in-depth analysis of the glycoproteoform expressed.

Previous research has focused on synthesis optimization of monolithic HILIC columns to improve separation of monoclonal antibody (mAb) samples. The current methods suffers from carryover requiring several blank injections between samples, and requires 50 ng to be loaded on-column.

This study systematically investigates methods to mitigate the carryover affects by employing self-packed PLRP-S stationary phase trap columns and changing washing solvents and modes. Additionally, we also explore the decrease in column I.D. as a method to decrease the flowrate and gain sensitivity, potentially allowing for analysis of sample loading of a few ng on-column. Our preliminary results show that the PLRP-S trap columns can reduce the carryover from the trap column due to a decrease in secondary interactions. We also observed increases in sensitivity when decreasing the I.D. of the HILIC columns and flowrate. The developed workflow allows for analysis of IgG glycan distributions with reduced carryover and increased sensitivity. In the next steps, we aim to apply this optimized method to the in-depth IgG glycoform characterization of exposed B-cells.