Category: Formulation and Quality
Purpose: Bispecific antibodies (bsAb) are a unique category of antibody therapeutics, which may have enhanced target specificity and efficacy compared with monoclonal antibodies. While bsAb therapeutics are often administered at a low dose by IV infusion, a high protein concentration formulation of a bsAb for subcutaneous injection may be needed depending on several factors, such as therapeutic target and indication, patient population and competitive landscape. However, research in high concentration formulation development of bsAb is limited. In this study, we characterized the high concentration behaviors of a bsAb and investigated the molecular mechanism of these behaviors by molecular modeling and characterizing the impact of formulation parameters, such as ionic strength and pH, on solution behaviors of bsAb and the associated monospecific monoclonal antibodies (mAbs).
Methods: In silico analysis - The homology models of proteins were built with MOE 2018. The pI value was calculated based on the static model structure using the protein properties analysis module. Surface properties were calculated with BioMOE module.
Biophysical characterization - Solution turbidity was measured by optical density (OD) at 405 nm. Apparent viscosity of protein solutions at various formulation conditions was measured by microchip-based viscometer. Interaction parameter kD was measured with dynamic light scattering (DLS). Second viral coefficient was measured by composition gradient-multi-angle light scattering (CG-MALS). Dimensionless retention time from HIC-HPLC elution time was calculated to rank order the relative surface hydrophobicity.
Results: When formulated in 10 mM histidine at pH 6.0, high opalescence and viscosity were observed for the bsAb even at moderate protein concentrations. The intermolecular interaction parameter kD analysis showed that significant attractive protein-protein interactions (PPIs) exist in the bsAb solution at 10 mM histidine pH 6.0. Addition of salts, such as 150 mM NaCl or 150 mM ArgHCl, and reducing pH from 6.0 to 5.0 can effectively reduce the opalescence and viscosity of BsAb. Interestingly, 150 mM ArgHCl was found to reduce protein viscosity and attractive PPIs to a greater extent as compared to 150 mM NaCl.
Considering the overall low protein charge at pH 6.0, the strong effect of ionic strength and pH on high concentration behaviors suggested that short range electrostatic interactions such as dipole‑dipole interaction are responsible for the attractive PPIs. The greater effect provided by ArgHCl as compared to NaCl indicated hydrophobic interaction also promotes attractive PPIs at high protein concentration. This is supported by the considerable hydrophobicity feature of the bsAb in HIC-HPLC analysis.
Surface analysis of the bsAb by molecular modeling shows significantly different surface properties such as charge and hydrophobicity in the two Fab arms. To further understand the contribution of each Fab arm of the bsAb in relation to the observed high concentration behaviors, the monospecific mAbs A and B were characterized. The results showed that the order of the significance in opalescence and viscosity was mAb A > bsAb > mAb B, and similar effects of salts (NaCl and ArgHCl) and pH on the solution behaviors were found in mAb A, but not mAb B. Furthermore, the level of PPIs of bsAb was approximate to the average of the monospecific mAbs across all formulation conditions. The viscosities of bsAb and the monospecific antibodies followed reasonably well with the Arrhenius mixing rule, suggesting there is minimal cross-interactions between the two arms of bsAb while one arm is dominating the self-interaction.
Conclusion: In conclusion we discovered that high concentration behaviors of the bsAb are attributed to short range electrostatic interaction and hydrophobic interaction, with one Fab arm dominating the attractive self-interaction. Therefore, high concentration behaviors can be effectively modulated by ionic strength, pH and addition of hydrophobic excipients. Our study also demonstrated that intermolecular interaction parameter kD can provide reasonable prediction for protein high concentration behaviors and it is practical to use kD in molecular candidate selection and formulation optimization.