The Effect of Arginine as an Anti-Aggregation Excipient on Recombinant Human Growth Hormone

Abstract

Aggregation is one of the main physical instabilities of proteins, which might occur during all steps of the manufacturing and storage of products. The presence of protein aggregates may result in the reduction of activity, induce immunologic responses and failure of therapeutic efficiency. Therefore, using additives in drug formulations is one of the essential approaches to prevent protein aggregation. The main objective of this study was to evaluate the inhibitory influence of arginine or glycine as excipients on the aggregation behavior of recombinant human growth hormone (rhGH). Two types of mechanical and thermal stresses including freeze-thaw and vortex-agitation were applied to the 1 mg/mL protein solution in PBS buffer (25 mM, pH = 7) in the presence and absence of arginine and glycine. The influence of arginine or glycine at the concentration of 320 mM on reduction of rhGH thermal/mechanical-induced aggregation was evaluated using SE-HPLC and turbidity measurement. The results of this study revealed that the monomer concentration decreased linearly; and therefore, aggregate formation was intensified with the increase in the number of freeze-thaw cycles. Moreover, it was found that a significant amount of rhGH (> 80%) was rapidly adsorbed at the walls of the vessels or converted to insoluble aggregates. Arginine decreased the insoluble aggregate formed during the freeze-thaw cycling more effectively than glycine. In addition, following the vortex-agitation stress, arginine had the optimum preventive effect in aggregate formation in contrast to glycine, which increased the formation of insoluble aggregates. The findings revealed that arginine may be a potential additive in preserving rhGH against thermal/mechanical-induced aggregation. Highlights Aggregation is one of the main physical instabilities of proteins. Protein aggregation may result in a compromise of safety and efficacy of biopharmaceutical products. Arginine at the concentration of 320 mM reduced rhGH thermal/mechanical-induced aggregation. Arginine, in contrast to glycine, optimally decreased the formation of insoluble aggregates.