Investigating protein–excipient interactions of a multivalent V(HH) therapeutic protein using NMR spectroscopy

Multispecific therapeutic proteins come in a variety of formats, including bi- and tri-specific antibodies, dual-variable domain antibodies, and CrossMabs. These multivalent proteins are engineered to interact with multiple therapeutic target proteins with high specificity. Multi-domain proteins can be created by linking together a variety of high-affinity antibody fragments. The choice of protein domains and linkers not only affects the interactions of these molecules with therapeutic targets but also influences the intrinsic behavior in solution that affects their stability. The complexity of solution interactions may translate into developability and manufacturing challenges. Here, we use nuclear magnetic resonance (NMR) spectroscopy to study the solution behavior of a multivalent V(HH) molecule composed of three flexibly linked heavy-chain-only domains that show dramatic stabilization against thermal degradation in the presence of sucrose. A collection of NMR fingerprinting and profiling methods were used to simultaneously monitor the protein solution behavior and capture details of protein–excipient interactions. We provide a framework to characterize and begin to understand the role of molecular flexibility in protein stabilization with potential applications in the design of novel therapeutic protein scaffolds that include multivalent proteins, fusion proteins, antibody-drug conjugates, and proteins modified with flexible lipids. ALPHABETICAL LIST OF ABBREVIATIONS: Fab Fragment antigen-binding; Fc Fragment crystallizable; HMW High molecular weight; ∆HMW Difference between HMW species at stress temperature and 5°C controls; IgG Immunoglobulin G; mAbs Monoclonal antibodies; MV-V(HH) Multivalent V(HH) molecule with the format aC-L(1)-aC-L(1)-aD; NMR Nuclear magnetic resonance; scFv Single-chain fragment variable; SEC Size-exclusion chromatography; V(HH) Variable domain of Heavy chain of Heavy chain-only antibody