Probing the Protein–Excipient Interaction in the Orally Delivered Protein by Solid-State Hydrogen–Deuterium Exchange Mass Spectrometry and Molecular Dynamics

The oral administration of protein therapeutics in solid dosage form is gaining popularity due to its benefits, such as improved medication adherence, convenience, and ease of use for patients compared to traditional parental delivery. However, formulating oral biologics presents challenges related to pH barriers, enzymatic breakdown, and poor bioavailability. Therefore, understanding the interaction between excipients and protein therapeutics in the solid state is crucial for formulation development. In this Letter, we present a case study focused on investigating the role of excipients in protein aggregation during the production of a solid dosage form of a single variable domain on a heavy chain (VHH) protein. We employed solid-state hydrogen–deuterium exchange coupled with mass spectrometry (ssHDX-MS) at both intact protein and peptide levels to assess differences in protein–excipient interactions between two formulations. ssHDX-MS analysis revealed that one formulation effectively prevents protein aggregation during compaction by blocking β-sheets across the VHH protein, thereby preventing β-sheet−β-sheet interactions. Spatial aggregation propensity (SAP) mapping and cosolvent simulation from molecular dynamics (MD) simulation further validated the protein–excipient interaction sites identified through ssHDX-MS. Additionally, the MD simulation demonstrated that the interaction between the VHH protein and excipients involves hydrophilic interactions and/or hydrogen bonding. This novel approach holds significant potential for understanding protein–excipient interactions in the solid state and can guide the formulation and process development of orally delivered protein dosage forms, ultimately enhancing their efficacy and stability.