Contributions of local mobility and degree of retention of native secondary structure to the stability of recombinant human growth hormone (rhGH) in glassy lyophilized formulations

Abstract

Recombinant human growth hormone (rhGH) was used as a model in lyophilized formulations to investigate how protein degradation pathways of aggregation, deamidation and oxidation are linked to glassy state dynamics and the structure of protein immobilized in glassy matrices. This study examined formulations of rhGH combined with the disaccharides sucrose or trehalose and various amounts of hydroxyethyl starch (HES), which upon lyophilization yielded glasses with a wide range of retention of native protein structure and glass transition temperatures. After storage at temperatures of 313, 323 and 333 K for periods of up to 16 weeks, samples were analyzed for protein secondary structure, the amount of various types of protein degradation, as well as structural relaxation time and the local mobility. Surprisingly, protein degradation rates were higher in formulations with higher glass transition temperatures and slower global dynamic motions. Formulations in which the protein retained more native-like structure immediately following lyophilization exhibited better stability, and these formulations also showed larger free energy barriers to structural perturbations. Protein structure and local mobility in the glass were strongly correlated with rate constants for aggregation, deamidation and oxidation local mobility within the glassy matrices, but degradation rates had different dependencies on these stability metrics at different temperatures.