Hemoglobin-based high-throughput assessment of lyoprotectant efficacy

Biologics, including vaccines, therapeutic proteins, blood products, and cell therapies, are susceptible to degradation during drying and therefore require the incorporation of protective additives (lyoprotectants) into the processing formulation to maintain their desired biological activities after reconstitution. However, assays to quantitatively assess lyoprotectant efficacy remain limited. A hemoglobin oxidation assay was developed into a high-throughput platform for screening the protective properties of compounds during freeze-drying. Hemoglobin, a redox-sensitive protein, oxidizes upon drying and rehydration, providing a robust reporter of desiccation-induced protein denaturation. Concentration-dependent protection by saccharides and polymers was quantified as the concentration required to inhibit 50% of hemoglobin oxidation (IC₅₀) after drying and rehydration. When comparing carbohydrates, raffinose exhibited the strongest protection on a molar basis (IC₅₀ = 2.5 ± 0.5 mM), but when expressed as mass-based concentration (% w/v), the IC₅₀ values showed little to no difference across carbohydrates (IC₅₀ = 0.12 ± 0.02 % w/v). To complement experimental results, all-atom molecular dynamics simulations of hemoglobin were employed in the presence of glucose, trehalose, and raffinose under hydrated and frozen conditions. Simulations demonstrated that larger carbohydrates form more hydrogen bonds with hemoglobin and accumulate more extensively on its surface, displacing hydration water and providing a protective "coat" during freezing. Together, these results implicate water replacement and surface coverage as principal mechanisms by which sugars stabilize biologics during drying, likely operating coordinately with vitrification. The integrated experimental-computational framework establishes a quantitative, mechanistically informed approach for evaluating, advancing discovery, and characterizing lyoprotectants, and for designing optimal stabilizers for freeze-dried red blood cells.