title: Conformational Analysis of Proteins in Highly Concentrated Solutions by Dialysis-Coupled Hydrogen/Deuterium Exchange Mass Spectrometry.
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, "traditional" 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. Graphical Abstract ᅟ.
authors: Protein Pharmaceutical Development, Biogen, Cambridge, MA, 02142, USA. damian.houde@biogen.com.
authors: Damian
authors: D
authors: Houde
authors: Department of Pharmacy, University of Copenhagen, Copenhagen, Denmark.
authors: Zeinab E
authors: ZE
authors: Nazari
authors: Protein Pharmaceutical Development, Biogen, Cambridge, MA, 02142, USA.
authors: George M
authors: GM
authors: Bou-Assaf
authors: Protein Pharmaceutical Development, Biogen, Cambridge, MA, 02142, USA.
authors: Andrew S
authors: AS
authors: Weiskopf
authors: Department of Pharmacy, University of Copenhagen, Copenhagen, Denmark.
authors: Kasper D
authors: KD
authors: Rand
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D016466:CHO Cells; D003412:Cricetulus; D041961:Deuterium Exchange Measurement / Q000295:instrumentation*; D003956:Dialysis / Q000295:instrumentation*; D004867:Equipment Design; D013058:Mass Spectrometry / Q000295:instrumentation*; D008958:Models, Molecular; D011487:Protein Conformation; D012996:Solutions
chemical_list: D000911:Antibodies, Monoclonal; D012996:Solutions
keywords: Dialysis; HDX; High concentration mAbs; Hydrogen deuterium exchange; Protein conformation