Elucidation of the Reversible Self-Association Interface of a Diabody–Interleukin Fusion Protein Using Hydrogen-Exchange Mass Spectrometry and In Silico Modeling

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.