HILIC-MS for biopharmaceutical analysis and (glyco)proteomics


Govert Somsen, Division of Bioanalytical Chemistry, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands (g.w.somsen@vu.nl)
Sara Tengattini, Department Of Drug Sciences And Italian Biocatalysis Center, University Of Pavia, Pavia, Italy
Thomas Senard, Center For Proteomics And Metabolomics, Leiden University Medical Center, Leiden, The Netherlands
Guusje van Schaick, Division Of Bioanalytical Chemistry, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands
Gestur Vidarsson, Department Of Experimental Immunohematology, Sanquin Research And Landsteiner Laboratory, Amsterdam, The Netherlands
Rob Haselberg, Division Of Bioanalytical Chemistry, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands
Manfred Wuhrer, Center For Proteomics And Metabolomics, Leiden University Medical Center, Leiden, The Netherlands
David Falck, Center For Proteomics And Metabolomics, Leiden University Medical Center, Leiden, The Netherlands
Elena Dominguez-Vega, Division Of Bioanalytical Chemistry, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands
Andrea Gargano, Division Of Bioanalytical Chemistry, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands

Liquid chromatographic (LC) methods that provide both efficient separation of intact proteins and compatibility with mass spectrometry (MS) are of essential importance in biopharmaceutical analysis and top-down proteomics. Recently, hydrophilic interaction liquid chromatography (HILIC) using amide stationary phases has shown highly suitable for analysis of intact proteins. This presentation outlines HILIC-MS methodologies for protein analysis highlighting the following main aspects:

- HILIC is suitable for the separation of a wide range of proteins, exhibiting a selectivity orthogonal to RPLC.

- Using appropriate eluent gradients, HILIC-MS shows a unique selectivity towards protein glycosylation, providing high resolution of intact glycoforms.

- Computer-aided method development facilitates and strongly accelerates optimization of glycoform separations by HILIC.

- Capillary HILIC-MS employing trap-columns for pre-concentration and dopant-gas for enhanced ionization substantially boosts sensitivity, allowing detection of low-abundant proteins from biological samples.

The attainable HILIC-MS performance will be illustrated by characterization of pharmaceutical proteins such as interferon-beta, erythropoietin, antigen glycoconjugates and monoclonal antibodies, but also by analysis of a complex cell lysate and detailed profiling of IgGs in human plasma.


Abstract Reference & Short Personal Biography of Presenting Author

Govert Somsen is full professor of Biomolecular Analysis/Analytical Chemistry at the Vrije Universiteit Amsterdam, The Netherlands. He obtained his doctorate in Amsterdam, and subsequently was assistant and associate professor at the University of Groningen and at Utrecht University in The Netherlands. His current research interests include the compositional and conformational characterization of intact biomacromolecules, and the bioactivity screening of compounds in complex samples. His group built an internationally recognized expertise in the hyphenation of separation techniques with mass spectrometry and optical spectroscopy for the analysis of intact proteins. Somsen is (co)author of over 185  peer-reviewed papers and has an H-index of 39. He is editor of Journal of Chromatography B.


[1] E. Domínguez-Vega, S. Tengattini, C. Peintner, J. van Angeren, C. Temporini, R. Haselberg, G. Massolini, G.W. Somsen, High-resolution glycoform profiling of intact therapeutic proteins by hydrophilic interaction chromatography-mass spectrometry, Talanta 184 (2018) 375-381.

[2] A.F.G. Gargano, L.S. Roca, R.T. Fellers, M. Bocxe, E. Domínguez-Vega, G.W. Somsen, Capillary HILIC-MS: a new tool for sensitive top-down proteomics, Anal. Chem. 90 (2018) 6601-6609.

[3] G. van Schaick, B.W. Pirok, R. Haselberg, G.W. Somsen, A.F.G. Gargano, Computer-aided gradient optimization of hydrophilic interaction liquid chromatographic separations of intact proteins and protein glycoforms, J. Chromatogr. A, in press, doi.org/ 10.1016/j.chroma.2019.03.038.

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