Abstract
Analytical methods that allow separation and identification of therapeutic proteins under native conditions play a crucial role in studying their higher-order structures and structure–function relationships. Recently, hyphenated techniques that combine native-mode separation with native mass spectrometry (nMS) have emerged as highly valuable tools for the targeted assessment of these quality attributes. This article outlines current native separation strategies coupled with nMS designed to characterize biopharmaceuticals close to their natural state. The methods provide worthwhile insights into aspects like aggregation, charge variants, conjugate stoichiometry, affinity, and conformation. As multidimensional chromatographic techniques and ion-mobility spectrometry become more accessible in laboratories, further advances in the development of native hyphenated techniques capable of simultaneously providing compositional, structural, and functional information on biopharmaceuticals can be expected.
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