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 a nd structure of the pharmaceutical protein recombinant human growth ho rmone (rhGH) in the lyophilized state. All excipients afforded signifi cant protection of the protein against aggregation, particularly at le vels 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 man nitol 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) spectrosc opy. rhGH exhibited a decrease in alpha-helix and increase in beta-she et structures upon drying. Addition of excipient stabilized the second ary structure upon lyophilization to a varying extent depending on the formulation. Samples with a significant degree of structural conserva tion, as indicated by the alpha-helix content, generally exhibited red uced aggregation. In addition, prevention of protein-protein interacti ons (indicated by reduced beta-sheet formation) also tended to result in lower rates of aggregation. Therefore, in addition to preserving th e protein structure, bulk additives that do not crystallize easily and remain amorphous in the solid state can be used to increase protein-p rotein distance and thus prevent aggregation.