Vacuum Induced Surface Freezing for the Freeze-Drying of the Human Growth Hormone: How does Nucleation Control Affect Protein Stability?

Abstract In the present work, the effect of controlled nucleation on the stability of human growth hormone (hGH) during freeze-drying has been investigated. More specifically, the vacuum induced surface freezing technique has been compared to conventional freezing, both with and without an annealing step. Size exclusion chromatography and cell-based potency assays have been used to characterize the formation of soluble aggregates and the biological activity of hGH, respectively. The results obtained indicate that controlled nucleation has a positive effect on both cycle performance and product homogeneity, because of the formation of bigger ice crystals, and characterized by a narrower dimensional distribution. From the point of view of hGH stability, we observed that vacuum induced surface freezing is not detrimental to the biological activity of the protein, or aggregate formation. Moreover, the effect of two different formulations, including trehalose or cellobiose, on protein preservation was also considered for this study.