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.

In this work we describe the structural investigation of the model protein recombinant human growth hormone (rhGH) under conditions relevant to polymeric sustained-delivery depots, including the dried protein entrapped in a film of poly(DL-lactic-co-glycolic)acid. At each step of the procedure, dehydration of rhGH by lyophilization, suspension in methylene chloride, and drying from that suspension, the structure of rhGH was probed noninvasively using Fourier transform infrared (FTIR) spectroscopy. We found that the structure of rhGH was significantly changed by the dehydration process as indicated by a marked drop in the alpha-helix content and increase in the beta-sheet content. Subsequent suspension of this powder in methylene chloride, drying from that suspension, and drying from a methylene chloride/PLGA solution introduced only minor additional structural changes when using appropriate conditions. This result is likely due to the limited molecular mobility of proteins in nonprotein-dissolving organic solvents. Finally, when rhGH was co-lyophilized with the lyoprotectant trehalose, which preserves the secondary structure, the rhGH entrapped in the PLGA matrix also had a nativelike secondary structure.

Acromegaly is a rare and chronic condition that is characterized by sustained unregulated hypersecretion of growth hormone (GH). More than 99% of the cases of acromegaly are due to a pathologic proliferation of pituitary somatotrophs presenting in the form of a pituitary adenoma. The excessive amounts of GH and its target hormone, insulin like growth factor-1 (IGF-1) cause metabolic changes and tissue enlargement that, collectively, lead to significant morbidity and a two to threefold increase in mortality. Thus, early diagnosis has proved to be crucial to improve survival and quality of life in this condition. The development of radioimmunoassay (RIA) in the 1960s provided clinicians with a biochemical tool to diagnose acromegaly. Many limitations were inherent to this methodology which necessitated the development of more sensitive tools, such as immunoradiometric (IRMA) or immunoluminometric (ILMA) assays for GH and IGF-1 measurements. These newer assays have not come without imperfections. The reference ranges to describe normalcy of the somatotropic axis and the biochemical criteria of "cure" of acromegaly are areas of great debate. Nevertheless, the current international consensus agrees that the diagnosis of acromegaly should be based on both clinical presentation and biochemical data.

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