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

Abstract

The use of tert-butyl alcohol for the lyophilization of pharmaceuticals has seen an uptick over the past years. Its advantages include increased solubility of hydrophobic drugs, enhanced product stability, shorter reconstitution time, and decreased processing time. While the mechanisms of protein stabilization exerted by cryo- and lyo-protectants are well known when water is the solvent of choice, little is known for organic solvents. This work investigates the interactions between two model proteins, namely, lactate dehydrogenase and myoglobin, and various excipients (mannitol, sucrose, 2-hydroxypropyl-β-cyclodextrin and Tween 80) in the presence of tert-butyl alcohol. We thermally characterized mixtures of these components by differential scanning calorimetry and freeze-drying microscopy. We also spectroscopically evaluated the protein recovery after freezing and freeze-drying. We additionally performed molecular dynamics simulations to elucidate the interactions in ternary mixtures of the herein-investigated excipients, tert-butyl alcohol and the proteins. Both experiments and simulations revealed that tert-butyl alcohol had a detrimental impact on the recovery of the two investigated proteins, and no combination of excipients yielded a satisfactory recovery when the organic solvent was present within the formulation. Simulations suggested that the denaturing effect of tert-butyl alcohol was related to its propensity to accumulate in the proximity of the peptide surface, especially near positively charged residues.

Keywords: cosolvent formulations; cyclodextrins; freeze-drying; molecular dynamics; protein stability; tert-butyl alcohol.

Figure 1

Snapshots of the simulated molecules realized with Visual Molecular Dynamics 1.9.3 (VMD).

Figure 2

Recovery of protein after fast (black columns) and slow (dashed columns) freeze/thawing or freeze-drying (white columns). According to ANOVA (p-value < 0.05), columns with different letters are statistically different. Myoglobin recovery as measured by OD at 280 nm (a) and at 410 nm (b). Lactate dehydrogenase recovery as measured by enzymatic activity (c).

Figure 3

β-Parameter profiles for the various formulations containing Mb in the liquid bulk, corresponding to simulations from 2b to 3d of Table 3. Left column: Mb parameterized with the CHARMM36m force field. Right column: Mb parameterized with the GROMOS 54a7 force field. The legend box indicates the excipients in solution, if any. Panels a and d: profiles for the excipients in bulk water, simulations 2b–2d. Panels b and e: profiles for the excipients in the 20% w/w TBA bulk solution, simulations 3b–3d. Panels c and f: profiles for TBA in the 20% w/w TBA bulk solution, simulations 3a-3d.