Complementary Ligand Binding Assay and Intact Mass Spectrometry Strategies for Preclinical Pharmacokinetics of Trispecific Antibody Candidates

Multispecific monoclonal antibodies present bioanalytical challenges for pharmacokinetic (PK) assessment because total concentration measurements alone do not resolve molecular integrity, stoichiometry, or product-related heterogeneity. Here, complementary ligand-binding assay (LBA) and intact liquid chromatography–mass spectrometry (LC-MS) methods were applied to a mouse PK study of a monospecific IgG1, a trispecific Variable Heavy-chain domain of a Heavy-chain antibody (VHH)-containing DuetmAb (Duet VHH4), and a trispecific monoclonal antibody (Trimab). LBA was used for quantitative plasma PK profiling, while an intact LC-MS workflow was implemented to assess circulating intact mass profiles, chain mispairing, and biotransformations. LBA quantified PK for all three molecules, with exposure rank order of Trimab > IgG1 > Duet VHH4. Intact LC-MS confirmed that the predominant circulating species remained structurally intact across the study and further resolved chain mispairing and glycation, features not directly observable by LBA and not preserved in digestion-based LC-MS workflows. Data presented herein demonstrate that intact LC-MS provides molecularly specific readouts complementary to LBA-derived exposure data and enables direct assessment of in vivo structural fidelity for trispecific antibody (TsAb) formats. The combined approach supports preclinical PK characterization and lead prioritization for complex TsAbs.