Effects of thermal and mechanical stress on the physical stability of human growth hormone and epidermal growth factor

Abstract

Thermal and mechanical stress conditions were applied to two model proteins, human growth hormone (hGH) and epidermal growth factor (EGF), to evaluate protein stability during the manufacturing process, focusing on protein secondary structure and aggregation. The samples were analyzed with differential scanning calorimetry (DSC), circular dichroism (CD), and size-exclusion chromatography (SEC). The monomer and aggregation contents were obtained by SEC and the proteins' secondary structure on exposure to thermal stress was evaluated by CD. DSC showed that the transition temperature (T (m)) of hGH and EGF was 74.43 and 79.11 A degrees C, respectively. The accelerated thermal stress temperature was set at 70 A degrees C. The monomer content of hGH decreased from 97.8 to 82.3 % in response to thermal stress. However, the monomer content of EGF decreased significantly from 33.73 to 5.61 %. The hGH and EGF showed an increase in alpha-helix content and a decrease in beta-sheet (antiparallel and parallel beta-sheet). Moreover, the contents changed significantly during the first 1 h and then changed slightly for the remaining time. On the other hand, shaking stress showed that hGH was highly affected compared to EGF. The hGH monomer steadily decreased and only the half the monomer content remained at 3 h. It is suspected that the shaking stress induced hGH adsorption to the gas-liquid interface, which may facilitate protein denaturation. The results indicate that protective excipients might be necessary for inevitable stress conditions during the developmental process. The stability of each protein differed with respect to specific stress conditions. Therefore, an array of complementary analytical methods might be required to evaluate the protein stability.