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Van 't Hoff Institute for Molecular Sciences Van 't Hoff Institute for Molecular Sciences

Advanced analysis of intact antibodies and protein complexes

10 July 2025

PhD candidates Ziran Zhai and Annika van der Zon of the Chemometrics & Advanced Separations group at the Van ‘t Hoff Institute for Molecular Sciences have developed advanced low-flow methods for the analysis of intact antibodies and protein complexes. In two recent papers, they present novel nanoSEC-MS and HILIC-MS methods that offer improved sensitivity and performance for bioanalysis.

The two PhD candidates work under the supervision of Andrea Gargano.

NanoSEC-MS for protein complexes

Ziran Zhai developed a novel nanoflow size exclusion chromatography–native mass spectrometry (nanoSEC-nMS) method and demonstrated its capability for analyzing minute amounts of intact protein complexes.

NanoSEC-nMS analysis of urine samples and Ovitrelle: (a) EIC of the urine hCG samples; (b) MS spectrum of hCG proteins; (c) deconvoluted results of hCG proteins; (d) EIC of the Ovitrelle sample; (e) MS spectrum of Ovitrelle; (f) deconvoluted results of Ovitrelle. Image: HIMS / Analytical Chemistry. Click to enlarge.

It enables the analysis of proteins and complexes across a broad molecular weight range (10 to 250 kDa) in their native states, preserving noncovalently bound metal ions.

The method includes techniques for preparing high-performance capillary SEC columns, and for optimizing injection to reduce peak widths. It also enables direct coupling of nanoflow SEC with native MS even in salt-rich environments. The nanoflow system features milder desolvation by electrospray ionization (ESI), preserving the native structures of proteins and complexes. Finally, the method requires limited sample volumes (approx. 100 nL per injection) and significantly enhances native MS throughput, enabling online desalting and oligomer separations within 25 minutes.

Paper details:
Ziran Zhai, Thomas Holmark, Annika A. M. van der Zon, Vasilis Tseliou, Francesco G. Mutti, Alina Astefanei, and Andrea F. G. Gargano: Nanoflow Size Exclusion Chromatography–Native Mass Spectrometry of Intact Proteoforms and Protein Complexes. Analytical Chemistry 2025 97 (23), 12241-12250 DOI: 10.1021/acs.analchem.5c01019

 

HILIC-MS for antibodies

Annika van der Zon developed a novel method for precise glycoform profiling of intact antibodies with HILIC-MS. Compared to traditional methods, it significantly enhances glycoform selectivity for intact antibodies, enabling a comprehensive characterization that is essential for bioanalytical applications.

Analysis of intact Trastuzumab at the intact level. Base Peak Chromatogram of the analysis and Extracted Ion Currents of selected glycoforms are shown. Image: HIMS / Analytical Chemistry. Click to enlarge.

The novel method features a lab-made acrylamide-based monolithic stationary phase, enhancing separation efficiency. It enables sensitive measurement of low-abundance glycoforms in the nanogram injection range and achieves baseline separations for single and double Fc glycosylation, as well as partial separations for glycoforms differing by a single glycan unit.

Paper details:
Annika A. M. van der Zon, LoÏs N. Hana, Huda Husein, Thomas Holmark, Ziran Zhai, and Andrea F. G. Gargano: Hydrophilic Interaction Chromatography HRMS with Acrylamide Monolithic Columns: A Novel Approach for Intact Antibody Glycoform Characterization. Analytical Chemistry 2025 97 (25), 13569-13576 DOI: 10.1021/acs.analchem.5c02033

 

See also

Research Andrea Gargano: Separation and Mass spectrometry (Chemometrics & Advanced Separations group)