Characterizing Non-covalent Protein Complexes Using Asymmetrical Flow Field-Flow Fractionation On-Line Coupled to Native Mass Spectrometry

Iro Konstantina Ventouri*, Wayne Chang, Florian Meier, Roland Drexel, Govert W. Somsen, Peter J. Schoenmakers, Bart de Spiegeleer, Rob Haselberg*, Alina Astefanei*

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

We report an online analytical platform based on the coupling of asymmetrical flow field-flow fractionation (AF4) and native mass spectrometry (nMS) in parallel with UV-absorbance, multi-angle light scattering (MALS), and differential-refractive-index (UV-MALS-dRI) detectors to elucidate labile higher-order structures (HOS) of protein biotherapeutics. The technical aspects of coupling AF4 with nMS and the UV-MALS-dRI multi-detection system are discussed. The “slot-outlet” technique was used to reduce sample dilution and split the AF4 effluent between the MS and UV-MALS-dRI detectors. The stability, HOS, and dissociation pathways of the tetrameric biotherapeutic enzyme (anticancer agent) l-asparaginase (ASNase) were studied. ASNase is a 140 kDa homo-tetramer, but the presence of intact octamers and degradation products with lower molecular weights was indicated by AF4-MALS/nMS. Exposing ASNase to 10 mM NaOH disturbed the equilibrium between the different non-covalent species and led to HOS dissociation. Correlation of the information obtained by AF4-MALS (liquid phase) and AF4-nMS (gas phase) revealed the formation of monomeric, tetrameric, and pentameric species. High-resolution MS revealed deamidation of the main intact tetramer upon exposure of ASNase to high pH (NaOH and ammonium bicarbonate). The particular information retrieved from ASNase with the developed platform in a single run demonstrates that the newly developed platform can be highly useful for aggregation and stability studies of protein biopharmaceuticals.

Original languageEnglish
Pages (from-to)7487-7494
Number of pages8
JournalAnalytical chemistry
Volume95
Issue number19
Early online date5 May 2023
DOIs
Publication statusPublished - 16 May 2023

Bibliographical note

Funding Information:
I.K.V. acknowledges the HOSAna project, which is funded by the Netherlands Organization for Scientific Research (NWO) in the framework of the Programmatic Technology Area PTA-COAST4 of the Fund New Chemical Innovations (project nr. 053.21.117).

Publisher Copyright:
© 2023 The Authors. Published by American Chemical Society.

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