Mass spectrometry-based methods in characterization of the higher order structure of protein therapeutics

Kaltashov, Igor A.; Bobst, Cedric E.; Pawlowski, Jake W.; Wang, Guanbo

doi:10.1016/j.jpba.2020.113169

Cited by 25 publications

(31 citation statements)

References 89 publications

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“…Discussions of the roles of mass spectrometry in structural analysis and characterization, i.e., structural proteomics, in which HDX MS has been included , were published. There were multiple more specific reviews of MS-based methods for structural biology, which included HDX MS, as applied to specific entities including macromolecular complexes − and protein interfaces as drug targets, amyloid fibrils, viral proteins, , G-protein coupled receptors, , vaccine development, and protein therapeutics . Several reviews compare and contrast HDX MS with footprinting methods such as covalent labeling, − and one review compared HDX MS and cryoEM .…”

Section: Review Of the Hdx Ms Reviewsmentioning

confidence: 99%

“…There were multiple more specific reviews of MS-based methods for structural biology, which included HDX MS, as applied to specific entities including macromolecular complexes 22−24 and protein interfaces as drug targets, 25 amyloid fibrils, 26 viral proteins, 27,28 Gprotein coupled receptors, 29,30 vaccine development, 31 and protein therapeutics. 32 Several reviews compare and contrast HDX MS with footprinting methods such as covalent labeling, 33−35 and one review compared HDX MS and cryoEM. 36 These combination-type reviews point again to the penetration of HDX MS applications into many other areas of science and away from isolation as an analytical curiosity.…”

Section: ■ Review Of the Hdx Ms Reviewsmentioning

confidence: 99%

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Developments in Hydrogen/Deuterium Exchange Mass Spectrometry

et al. 2020

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Section: Review Of the Hdx Ms Reviewsmentioning

confidence: 99%

Section: ■ Review Of the Hdx Ms Reviewsmentioning

confidence: 99%

Developments in Hydrogen/Deuterium Exchange Mass Spectrometry

et al. 2020

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“…In both cases, the equilibrium between the two conformations is fast on the chromatographic time scale, and the distinct elution windows of the two forms of the protein reflect the influence exerted by the extent of haptenation on the equilibrium between the closed (nativelike) and open (partially disordered) conformations. Analysis of protein ion charge state distributions in native MS has become a commonly accepted tool in the analysis of conformational integrity of therapeutic proteins, 23 although its application to other biopharmaceutical products (especially those exhibiting high degrees of structural heterogeneity, such as haptenated carrier proteins) remains limited. Supplementing ESI MS measurements with limited charge reduction in the gas phase not only provides an elegant way toward evaluating the extent of CRM197 haptenation but also allows the emergence of bimodal charge state distribution in the ESI mass spectra to be observed, thereby enabling detection of conformational transitions within the carrier protein that have been previously demonstrated to be important attenuators of the vaccine's functional response.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Simultaneous Evaluation of a Vaccine Component Microheterogeneity and Conformational Integrity Using Native Mass Spectrometry and Limited Charge Reduction

Bobst

Sperry

Friese

et al. 2021

J. Am. Soc. Mass Spectrom.

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Analytical characterization of extensively modified proteins (such as haptenated carrier proteins in synthetic vaccines) remains a challenging task due to the high degree of structural heterogeneity. Native mass spectrometry (MS) combined with limited charge reduction allows these obstacles to be overcome and enables meaningful characterization of a heavily haptenated carrier protein CRM197 (inactivated diphtheria toxin conjugated with nicotine), a major component of a smoking cessation vaccine. The extensive conjugation results in a near-continuum distribution of ionic signal in electrospray ionization (ESI) mass spectra of haptenated CRM197 even after size-exclusion chromatographic fractionation. However, supplementing the ESI MS measurements with limited charge reduction of ionic populations selected within narrow m/z windows gives rise to well-resolved charge ladders, from which both masses and charge states of the ionic species can be readily deduced. Application of this technique to a research-grade material of CRM197/H7 conjugate not only reveals its marginal conformational stability (manifested by the appearance of high charge-density ions in ESI MS) but also establishes a role of the extent of haptenation as a major factor driving the loss of the higher order structure integrity. The unique information provided by native MS used in combination with limited charge reduction provides a strong argument for this technique to become a standard/required tool in the analytical arsenal in the field of biotechnology and biopharmaceutical analysis, where protein conjugates are becoming increasingly common.

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“…Mass spectrometry has recently emerged as a powerful tool for the characterization of biotherapeutics and their interactions with antigens. 36 , 37 In this regard, hydrogen-deuterium exchange mass spectrometry (HDX-MS) has proven its merit. This technology provides detailed insights into protein interactions 38–41 and is well-suited for the assignment of both linear and conformational epitopes.…”

Section: Introductionmentioning

confidence: 99%