Excipient effect on phenol-induced precipitation of human growth hormone and bovine serum albumin

Abstract

The aim of this study was to investigate the impact of phenol on the precipitation of bovine serum albumin (BSA) and human growth hormone (hGH), in the presence of other excipients frequently used in biological drugs for parenteral delivery. The focus of the study lieson incompatibilities observed in multidose formulations containing non-ionic surfactants and preservatives. Previous research has shown that above a critical concentration, phenol reduces the cloud point of polysorbate surfactants to room temperature or lower. Here, it is demonstrated that for BSA-polysorbate solutions, phenol-induced incompatibility is primarily controlled by this depression of the surfactant cloud point, resulting in turbidity and/or precipitation. However, for formulations with human growth hormone (hGH) in isotonic salt solutions, the precipitation mechanism is instead driven by protein-phenol interactions. The precipitation is affected by the concentration of sodium chloride and at low salt concentrations the incompatibility is again controlled by depression of the surfactant cloud point. The concentration of salt needed for protein induced precipitation seems to follow the Hofmeister series, with sodium chloride and sodium sulphate inducing precipitation at a lower salt concentration than sodium nitrate. Notably, non-ionic tonicity agents, such as glucose and mannitol, which are known to impact the surfactant cloud point depression of phenol, do not induce precipitation of hGH in the presence of phenol. In the system containing polysorbate, phenol and hGH, salt-triggered protein precipitation occurs at slightly higher sodium chloride concentrations than in solutions without polysorbate. This indicates a stabilizing effect of polysorbate on hGH below the cloud point. However, the stabilising effect is surfactant dependent, and in the presence of dodecyl maltoside, hGH precipitation occurs at much lower sodium chloride concentrations than for solutions with polysorbates. This illustrates the complexity of the interplay of excipients with each other and with the active ingredient (the protein) in the development of multidose pharmaceutics.