Optimizing colloidal stability and viscosity of multispecific antibodies at the drug discovery-development interface: a systematic predictive case study

The discovery and development of multispecific antibodies present unique challenges in optimizing their physicochemical properties to enhance developability and manufacturability. Common developability challenges include increased risk of aggregation, high viscosity, poor solubility, low expression yields, complex purification requirements, greater propensity for fragmentation, immunogenicity, or pharmacokinetics. In this study, we systematically investigate the solution behavior of engineered bispecific IgG1-VHH constructs derived from a parental NKp30 ×EGFR natural killer cell engager (NKCE) molecule, focusing on colloidal stability, hydrophobicity, thermal stability, pH sensitivity, and viscosity. By combining <i>in silico</i> predictions and experimental evaluations, we engineered variants with altered isoelectric points (pIs) of the Fab and VHH domains and characterized them across a broad, formulation-relevant pH range (pH 4.5-8.0). Our findings indicate that aligning slightly basic pI profiles (approximately 7.5-9.0) across variable domains within bispecific antibodies can effectively mitigate charge asymmetries in standard acidic formulations that may lead to unfavorable solution behavior. Importantly, rational design and early-stage experimental validation yielded optimized variants exhibiting significantly improved colloidal stability and viscosity compared to the starting molecule. This systematic study, the first of its kind for bispecific antibodies, highlights the value of integrating domain-level <i>in silico</i> assessments early in antibody design, facilitating efficient optimization toward improved solution behavior of multispecific biotherapeutics.