Effect of excipients on the stability and structure of lyophilized recombinant human growth hormone.

We have investigated the effect of mannitol, sorbitol, methyl alpha-D-mannopyranoside, lactose, trehalose, and cellobiose on the stability and structure of the pharmaceutical protein recombinant human growth hormone (rhGH) in the lyophilized state. All excipients afforded significant protection of the protein against aggregation, particularly at levels to potentially satisfy water-binding sites on the protein in the dried state (i.e., 131:1 excipient-to-protein molar ratio). At higher excipient-to-protein ratios, X-ray diffraction studies showed that mannitol and sorbitol were prone to crystallization and afforded somewhat less stabilization than at lower ratios where the excipient remained in the amorphous, protein-containing phase. The secondary structure of rhGH was determined using Fourier transform infrared (FTIR) spectroscopy. rhGH exhibited a decrease in alpha-helix and increase in beta-sheet structures upon drying. Addition of excipient stabilized the secondary structure upon lyophilization to a varying extent depending on the formulation. Samples with a significant degree of structural conservation, as indicated by the alpha-helix content, generally exhibited reduced aggregation. In addition, prevention of protein-protein interactions (indicated by reduced beta-sheet formation) also tended to result in lower rates of aggregation. Therefore, in addition to preserving the protein structure, bulk additives that do not crystallize easily and remain amorphous in the solid state can be used to increase protein-protein distance and thus prevent aggregation.