Mechanistic Investigation of tert-Butanol’s Impact on Biopharmaceutical Formulations: When Experiments Meet Molecular Dynamics

The use of tert-butyl alcohol for thelyophilizationof pharmaceuticals has seen an uptick over the past years. Its advantagesinclude increased solubility of hydrophobic drugs, enhanced productstability, shorter reconstitution time, and decreased processing time.While the mechanisms of protein stabilization exerted by cryo- andlyo-protectants are well known when water is the solvent of choice,little is known for organic solvents. This work investigates the interactionsbetween two model proteins, namely, lactate dehydrogenase and myoglobin,and various excipients (mannitol, sucrose, 2-hydroxypropyl-& beta;-cyclodextrinand Tween 80) in the presence of tert-butyl alcohol.We thermally characterized mixtures of these components by differentialscanning calorimetry and freeze-drying microscopy. We also spectroscopicallyevaluated the protein recovery after freezing and freeze-drying. Weadditionally performed molecular dynamics simulations to elucidatethe interactions in ternary mixtures of the herein-investigated excipients, tert-butyl alcohol and the proteins. Both experiments andsimulations revealed that tert-butyl alcohol hada detrimental impact on the recovery of the two investigated proteins,and no combination of excipients yielded a satisfactory recovery whenthe organic solvent was present within the formulation. Simulationssuggested that the denaturing effect of tert-butylalcohol was related to its propensity to accumulate in the proximityof the peptide surface, especially near positively charged residues.