Aggregation of recombinant human growth hormone induced by phenolic compounds

Phenolic compounds can be used as antimicrobial preservative agents in pharmaceutical formulations. Unfortunately, these compounds often adversely affect proteins, triggering aggregation in particular. In this study, a variety of phenolic compounds and structurally similar non-aromatic alcohols were investigated for their role in causing the aggregation of a model therapeutic protein, recombinant human growth hormone (rhGH). As determined by various methodologies, most of the phenolic compounds caused rhGH aggregation, especially at high concentrations. Stress studies under freezing, high-temperature incubation, and agitation suggest that the destabilizing influence of the compounds tested increases in the order of benzyl alcohol < phenol × resorcinol < catechol < meta-cresol < 2-chlorophenol. Non-aromatic alcohols, except 2,6-dimethylcyclohexanol, have a much less adverse effect. Determination of the thermal transition temperature by microcalorimetry studies also reflected this trend. From our study, we conclude: (1) the phenolic additive-induced rhGH aggregates were held by non-covalent forces; (2) no significant physical binding occurred between the protein and these compounds; (3) the aggregation tendency of the phenolic compounds failed to correlate with their hydrogen bonding strength; (4) the presence of a phenolic additive caused conformational changes in rhGH's structure; (5) the effect of these phenolic compounds on rhGH aggregation decreased at high and low pHs.