Monoclonal antibodies (mAbs) have become the largest class of biological drugs. Due to their inherently low bioavailability when administered orally, they must be administered parenterally with subcutaneous (SC) administration preferred for its potential for self administration. However, the volume that can be injected subcutaneously is limited, while therapeutic doses of mAbs are high. This necessitates highly concentrated formulations, which are often limited by high viscosity and low stability. In this study, we evaluated the influence of viscosity-reducing test compounds on the colloidal stability of highly concentrated mAb formulations. Using dynamic and static light scattering methods, we measured the diffusion interaction parameter (kD) and the second virial coefficient (B22) to evaluate protein-protein interactions in the formulation. While optimizing sample preparation, we evaluated the influence of pH, type of dilution medium, concentration of histidine and sodium chloride on the measured parameters. Phosphate buffer (pH 6.0) was chosen as it had minimal impact on kD and B22 values, while also matching the pH of the original highly concentrated formulation. The addition of sodium chloride to the dilution medium showed that kD increases with the ionic strength of the solution, likely by shielding repulsive electrostatic interactions. Therefore, we added 150 mM sodium chloride to the dilution medium to highlight the attractive interactions dominating in highly concentrated formulations. Then, we evaluated proline analogues as test compounds with viscosity-reducing effect. Increasing concentration of these test compounds (50, 100 and 200 mM) showed that kD and B22 values were highest in formulations at 200 mM, confirming that these compounds reduce the viscosity of the formulation by preventing mAb interactions. Combinations of test compounds were then evaluated. Based on kD results, we concluded that the test compound combinations (S32-10, S32 13, S32 15, and S36 17), each containing the respective compound at a concentration of 25 mM, reduced attractive protein-protein interactions, likely resulting in more stable formulations. Combinations proved to be a better strategy for reducing formulation viscosity, as they had less effect on mAb stability than higher concentrations of individual compounds. Nevertheless, kD and B22 values failed to differentiate between the test compounds in terms of their impact on interactions and stability, possibly due to the low concentration of the test compounds in the formulations.
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