%0 Journal Article
%T Elucidation of the Reversible Self-Association Interface of a Diabody-Interleukin Fusion Protein Using Hydrogen-Exchange Mass Spectrometry and In Silico Modeling.
%A Eisinger M
%A Rahn H
%A Chen Y
%A Fernandes M
%A Lin Z
%A Hentze N
%A Tavella D
%A Moussa EM
%J Mol Pharm
%V 0
%N 0
%D 2024 Jun 26
%M 38922328
%F 5.364
%R 10.1021/acs.molpharmaceut.4c00169
%X Reversible self-association (RSA) of therapeutic proteins presents major challenges in the development of high-concentration formulations, especially those intended for subcutaneous administration. Understanding self-association mechanisms is therefore critical to the design and selection of candidates with acceptable developability to advance to clinical trials. The combination of experiments and in silico modeling presents a powerful tool to elucidate the interface of self-association. RSA of monoclonal antibodies has been studied extensively under different solution conditions and have been shown to involve interactions for both the antigen-binding fragment and the crystallizable fragment. Novel modalities such as bispecific antibodies, antigen-binding fragments, single-chain-variable fragments, and diabodies constitute a fast-growing class of antibody-based therapeutics that have unique physiochemical properties compared to monoclonal antibodies. In this study, the RSA interface of a diabody-interleukin 22 fusion protein (FP-1) was studied using hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) in combination with in silico modeling. Taken together, the results show that a complex solution behavior underlies the self-association of FP-1 and that the interface thereof can be attributed to a specific segment in the variable light chain of the diabody. These findings also demonstrate that the combination of HDX-MS with in silico modeling is a powerful tool to guide the design and candidate selection of novel biotherapeutic modalities.