Mass spectrometry‐based methods to characterize highly heterogeneous biopharmaceuticals, vaccines, and nonbiological complex drugs at the intact‐mass level

Abstract

The intact‐mass MS measurements are becoming increasingly popular in characterization of a range of biopolymers, especially those of interest to biopharmaceutical industry. However, as the complexity of protein therapeutics and other macromolecular medicines increases, the new challenges arise, one of which is the high levels of structural heterogeneity that are frequently exhibited by such products. The very notion of the molecular mass measurement loses its clear and intuitive meaning when applied to an extremely heterogenous system that cannot be characterized by a unique mass, but instead requires that a mass distribution be considered. Furthermore, convoluted mass distributions frequently give rise to unresolved ionic signal in mass spectra, from which little‐to‐none meaningful information can be extracted using standard approaches that work well for homogeneous systems. However, a range of technological advances made in the last decade, such as the hyphenation of intact‐mass MS measurements with front‐end separations, better integration of ion mobility in MS workflows, development of an impressive arsenal of gas‐phase ion chemistry tools to supplement MS methods, as well as the revival of the charge detection MS and its triumphant entry into the field of bioanalysis already made impressive contributions towards addressing the structural heterogeneity challenge. An overview of these techniques is accompanied by critical analysis of the strengths and weaknesses of different approaches, and a brief overview of their applications to specific classes of biopharmaceutical products, vaccines, and nonbiological complex drugs.

Characterization of Large Immune Complexes with Size Exclusion Chromatography and Native Mass Spectrometry Supplemented with Gas Phase Ion Chemistry

“…The spectrum features several distinct clusters of the ionic signals representing APP2/mAb complexes with specific stoichiometry. Although the charge state distributions within each cluster are at least partially discernible, the charge state assignment can be problematic – particularly for ions at high m / z range – due to the peak broadening caused by incomplete ion desolvation in the gas phase . This is caused by the apparent increase in the extent of nonspecific adduct formation with the decrease of the protein ion charge states in native MS, a phenomenon that results in a monotonic mass shift ( m ) across the range of charges ( z ) within the protein ion charge state distribution in native MS .…”

“…Although the charge state distributions within each cluster are at least partially discernible, the charge state assignment can be problematic − particularly for ions at high m/z range − due to the peak broadening caused by incomplete ion desolvation in the gas phase. 32 This is caused by the apparent increase in the extent of nonspecific adduct formation with the decrease of the protein ion charge states in native MS, a phenomenon that results in a monotonic mass shift (m) across the range of charges (z) within the protein ion charge state distribution in native MS. 14 We approach this problem by using limited charge reduction, 14 which gives rise to well-defined charge ladders, from which both ionic mass and charge could be readily determined. The ability of a quadrupole mass filter to carry out ionic selection at high m/z (up to 20,000 in our case) allows the entire m/z region of interest to be covered with the limited charge reduction measurements (Figure 3).…”

Structural Characterization of a Pathogenic Antibody Underlying Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT)

“…The anti-PF4 IgG molecules extracted from a VITT patient’s blood were subjected to intact mass analysis under near-native conditions in solution and aggressive desolvation in the gas phase. This allowed the spectral crowding (typically observed in the low m / z regions of the mass spectra of highly heterogeneous proteins acquired under denaturing conditions , ) to be avoided while minimizing the presence of the signal corresponding to noncovalent adduct ions . Nevertheless, the anti-PF4 antibody mass spectrum is highly convoluted (Figure A), and its deconvolution without applying any restrictions yields ambiguous results.…”

“…This allowed the spectral crowding (typically observed in the low m/z regions of the mass spectra of highly heterogeneous proteins acquired under denaturing conditions 26,27 ) to be avoided while minimizing the presence of the signal corresponding to noncovalent adduct ions. 28 Nevertheless, the anti-PF4 antibody mass spectrum is highly convoluted (Figure 1A), and its deconvolution without applying any restrictions yields ambiguous results. To resolve this ambiguity, the limited charge reduction 29 was applied to multiple ionic populations selected within narrow m/z windows, revealing the presence of three distinct components (Figure 1B).…”

Probing the Architecture of Multisubunit Protein Complexes with In-line Disulfide Reduction and Native MS Analysis

“…Native mass spectrometry (MS) remains one of the most popular tools in the arsenal of biophysical techniques that are commonly used for characterization of noncovalent biopolymer assemblies. 1 While the level of detail provided by native MS cannot rival that of the high-resolution techniques (such as NMR, cryo-EM, and X-ray crystallography), it offers important advantages, such as minimal sample requirement, generous upper-mass limits, 2 notable tolerance to structural heterogeneity, 3 and the ability to work with relatively low-affinity and metastable complexes. 4 The mass measurements in native MS provide information only on the composition and stoichiometry of the noncovalent complexes; however, its capabilities may be further expanded by supplementing this technique with controlled fragmentation of the noncovalent assemblies in the gas phase to yield information on their architecture.…”