Scientific Abstract

Biotherapeutic-Target Binding Stoichiometry and Site Elucidation by Combining Native-MS and HDX-MS Approaches (WCBP)

Scientific Event

January 28-31, 2019
Washington D.C.


Peter Li, Annie He, Serah Liu, Emma Zhang, Chen Li, Wanlu Qu, Dongdong Wang, Jennifer S. Chadwick, Shiaw-Lin Wu
BioAnalytix Inc., Cambridge, MA.


HDX-MS has become an important tool for higher-order structure analysis and comparability assessments biologic drugs. When combined with recently developed Native Mass Spectrometry (Native MS), these approaches may be applied to further determine binding ratios (stoichiometry) and elucidate related binding regions (amino acid sequences), for such biotherapeutics with their targets. In this study, a bi-specific monoclonal antibody (mAb) and its target receptor antigen (Ag) were first analyzed using Native-MS to determine the stoichiometry of the mAb-Ag binding complex. Initially, the bi-specific mAb with target Ag ratio of 1:1 was determined by the Native MS, demonstrating the bi-specific nature of the biologic as binding only one of the 2 monovalent arms. HDX-MS was further used to locate the binding regions at Ag by elucidating differential deuterium (D uptake) region by region.  Important in the analysis, the heavily N- / O-glycosylated Ag was able to be profiled by HDX-MS, with the glycosylation elements not affecting the analysis, as is often a challenge for other techniques such as typical x-ray crystallography. In the HDX-MS analysis of the mAb-Ag complex, three distinct regions of Ag were observed as having substantially different D-uptake profiles, which could be correlated to a region with rigid binding, a region with less rigid binding and a region undergoing conformational change when bound to the mAb. This study shows how a combination of Native MS and HDX-MS can be used to effectively determine mAb-Ag complex stoichiometry and elucidate the related complex binding regions. Further, types of protein aggregation or protein-drug interactions could be determined as covalent or non-covalent using the approaches as well, for example with covalent aggregation being determined from a related peptide mapping analysis of the covalently linked sites, whereas non-covalent aggregation could be inferred from the similar Native and HDX-MS analysis approach for assessing stoichiometry (i.e. dimer, trimer or larger aggregates) and aggregation regions (i.e at CDR region or not). In this regard, interactions between protein therapeutics and small molecule drugs (protein-drug complexes) could be elucidated as covalent or non-covalent in nature with stoichiometry and site elucidation by the same approach as well. These approaches may have significant applicability both in early-stage biologic candidate optimization and selection and in later stage protein aggregation and protein-drug interaction characterizations.

View Our Featured Resources