"The More, the Better?": The Impact of Sugar-to-Protein Molar Ratio in Freeze-Dried Monoclonal Antibody Formulations on Protein Stability.

Abstract

Lyophilization is widely used to ensure the stability of protein drugs by minimizing chemical and physical degradation in the dry solid state. To this end, proteins are typically formulated with sugars that form an amorphous immobilizing matrix and stabilize hydrogen bonds replacing water molecules. The optimal amount of sugar required and protein stability at low excipient-to-protein molar ratios are not well understood. We investigated this by focusing on the physical stability of formulations, reflecting highly concentrated monoclonal antibody (mAb) lyophilizates at low sucrose to mAb ratios between 25:1 and 360:1. Additionally, the impact of different excipient types, buffer concentrations, and polysorbates was studied. The mAb stability was evaluated over up to three months at 25 and 40 °C. We investigated the "the more, the better" approach regarding excipient usage in protein formulation and the existence of a potential stabilizing threshold. Our findings show efficient monomeric content preservation even at low molar ratios, which could be explained based on the water replacement theory. We identified an exponential correlation between the sucrose to protein molar ratio and aggregate formation and found that there is no molar ratio threshold to achieve minimum stabilization. Sucrose demonstrated the best stabilization effect. Both mannitol, used as a cryoprotectant at low concentrations, and arginine reduced aggregation compared to the pure mAb formulation. The higher ionic strength of 5 mM histidine buffer enhanced protein stability compared to that of 0.1 mM histidine buffer, which was more pronounced at lower molar ratios. The addition of polysorbate 20 contributed an additional interfacial stabilizing effect, complementing the cryoprotective and lyoprotective properties of sucrose. Overall, a model could be developed to optimize the quantity of sugar required for protein stabilization and facilitate a more rational design of protein lyophilizates. The molar ratio of sugar to protein for high-concentration mAb products is limited by the acceptable tonicity, but we showed that sufficient stabilization can be achieved even at low molar ratios.