Formulation and stability of freeze-dried proteins: effects of moisture and oxygen on the stability of freeze-dried formulations of human growth hormone.

This research presents the results of a series of stability studies on freeze-dried formulations of human growth hormone (hGH). Chemical decomposition via methionine oxidation and asparagine deamidation as well as irreversible aggregation are characterized by HPLC. Water sorption isotherms, DSC thermograms, and pulsed proton NMR data are also obtained. No glass transition temperatures are observed in the temperature range of the stability studies. The pulsed NMR data suggest onset of greater mobility in the solid at a water content slightly higher than BET "monolayer" level. Stability of freeze-dried solids at 25 degrees C and 40 degrees C is studied as a function of residual moisture and exposure to oxygen. Formulations with and without a glycine/mannitol excipient system are studied. Significant levels of chemical decomposition and irreversible aggregation occur under most conditions with the effects of residual water content and "headspace oxygen" strongly dependent on the formulation. At low water content with minimal oxygen in the vial headspace, the glycine/mannitol formulation yields optimum stability. However, for either high water content or high oxygen content in the vial, stability of hGH without excipients is superior. The qualitative effect of residual moisture on stability depends on the temperature of the stability study. Generally, the stability of a sample adjusted to a given water content by desorption (during freeze-drying) is identical to the stability of a sample prepared by sorption of water on to a previously highly dried sample.