title: Investigating monoclonal antibody aggregation using a combination of H/DX-MS and other biophysical measurements.
abstract: To determine how structural changes in antibodies are connected with aggregation, the structural areas of an antibody prone to and/or impacted by aggregation must be identified. In this work, the higher-order structure and biophysical properties of two different monoclonal antibody (mAb) monomers were compared with their simplest aggregated form, that is, dimers that naturally occurred during normal production and storage conditions. A combination of hydrogen/deuterium exchange mass spectrometry and other biophysical measurements was used to make the comparison. The results show that the dimerization process for one of the mAb monomers (mAb1) displayed no differences in its deuterium uptake between monomer and dimer forms. However, the other mAb monomer (mAb2) showed subtle changes in hydrogen/deuterium exchange as compared with its dimer form. In this case, differences observed were located in specific functional regions of the CH 2 domain and the hinge region between CH 1 and CH 2 domains. The importance and the implications of these changes on the antibody structure and mechanism of aggregation are discussed.
authors.affiliation: Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts, 02115.
authors.forename: Roxana E
authors.initials: RE
authors.lastname: Iacob
authors.forename: George M
authors.initials: GM
authors.lastname: Bou-Assaf
authors.forename: Lee
authors.initials: L
authors.lastname: Makowski
authors.forename: John R
authors.initials: JR
authors.lastname: Engen
authors.forename: Steven A
authors.initials: SA
authors.lastname: Berkowitz
authors.forename: Damian
authors.initials: D
authors.lastname: Houde
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D016466:CHO Cells; D002152:Calorimetry, Differential Scanning; D003412:Cricetulus; D004220:Disulfides / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry; D008958:Models, Molecular; D011487:Protein Conformation; D055503:Protein Multimerization; D055550:Protein Stability; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction
chemical_list: D000911:Antibodies, Monoclonal; D004220:Disulfides
keywords: Antibody dimerization; Calorimetry (DSC); Chromatography; Domain swapping; Electrophoresis; Hydrogen-deuterium exchange mass spectrometry (H/DX-MS); Protein aggregation; Protein structure; Small-angle X-ray solution scattering (SAXS)