Conformational Analysis of Proteins in Highly Concentrated Solutions by Dialysis-Coupled Hydrogen/Deuterium Exchange Mass Spectrometry

Abstract

Abstract. When highly concentrated, an antibody solution can exhibit unusual behaviors, which can lead to unwanted properties, such as increased levels of protein aggregation and unusually high viscosity. Molecular modeling, along with many indirect biophysical measurements, has suggested that the cause for these phenomena can be due to short range electrostatic and/or hydrophobic protein-protein interactions. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a useful tool for investigating protein conformation, dynamics, and interactions. However, Btraditional^continuous dilution labeling HDX-MS experiments have limited utility for the direct analysis of solutions with high concentrations of protein. Here, we present a dialysis-based HDX-MS (di-HDX-MS) method as an alternative HDX-MS labeling format, which takes advantage of passive dialysis rather than the classic dilution workflow. We applied this approach to a highly concentrated antibody solution without dilution or significant sample manipulation, prior to analysis. Such a method could pave the way for a deeper understanding of the unusual behavior of proteins at high concentrations, which is highly relevant for development of biopharmaceuticals in industry.

Charge-mediated Fab-Fc interactions in an IgG1 antibody induce reversible self-association, cluster formation, and elevated viscosity

“…33 Mass spectrometry coupled to HX (HX-MS), extends the HX technique to complex, multi-domain macromolecules like mAbs. [37][38][39][40][41][42][43] Recently, we described a novel HX-MS method to map protein interfaces formed between mAbs undergoing reversible protein-protein interactions directly at up to 60 g/L. 39 Here, we applied this technique to investigate the molecular mechanism by which an IgG1 mAb (mAb-J) undergoes reversible self-association, and further probed this mechanism by a variety of other biophysical techniques.…”

Characterization of Excipient Effects on Reversible Self-Association, Backbone Flexibility, and Solution Properties of an IgG1 Monoclonal Antibody at High Concentrations: Part 1

“…[28][29][30][31][32][33][34][35][36] Hydrogen exchange-mass spectrometry (HX-MS) is a wellestablished high-resolution technique for exploring protein dynamics and conformational flexibility including the effect of excipients on aggregation propensity and RSA in low and high concentration mAb formulations. 13,[32][33][34][35][36][37][38] Under certain circumstances, such as RSA or interaction with excipients, the exchange rate can be altered in specific regions, indicating local alterations of backbone dynamics. 13 HX-MS has been used to elucidate molecular effects of excipients [32][33][34][35][36][37][38][39] and chemical changes (e.g., asparagine deamidation, 40 deglycosylation, 35 posttranslational modifications, 41 and engineered point mutations 42 ) on mAb conformational dynamics.…”

“…13,[32][33][34][35][36][37][38] Under certain circumstances, such as RSA or interaction with excipients, the exchange rate can be altered in specific regions, indicating local alterations of backbone dynamics. 13 HX-MS has been used to elucidate molecular effects of excipients [32][33][34][35][36][37][38][39] and chemical changes (e.g., asparagine deamidation, 40 deglycosylation, 35 posttranslational modifications, 41 and engineered point mutations 42 ) on mAb conformational dynamics. We recently demonstrated that a particular IgG1 (mAb-J) undergoes extensive RSA at high protein concentrations via electrostatic attractive interactions between segments of the Fab and Fc domains as determined by HX-MS analysis.…”

Hydrogen/deuterium exchange-mass spectrometry analysis of high concentration biotherapeutics: application to phase-separated antibody formulations

“…Recently, HDX-MS workflows designed for the analysis of high concentration protein samples have been described. 20,21 For example, a recently described HDX-MS methodology that relies upon reconstituting lyophilized mAb powders in a deuterated buffer was able to characterize mAb structures at 60 mg/mL. 20 This approach identified protein-protein interfaces associated with a concentration-dependent reversible selfassociation.…”

“…To overcome these limitations, a dialysis-coupled HDX-MS strategy was recently reported for mAb analysis, in which passive dialysis microcassettes are used for HDX labeling. 21 While this approach successfully sampled high concentration (200 mg/ mL) IgG4 formulations for comparison with low concentration (3 mg/mL) samples, the long timescales needed for dialysis likely render many known modes of protein motion inaccessible to the technology.…”