Department of Pharmaceutics, School of Pharmacy, University of Connecticut, Storrs, Connecticut 06269, USA.
J Pharm Sci. 2010 Feb;99(2):683-700. doi: 10.1002/jps.21960.
The objective of this research was to investigate the impact of thermal treatment on storage stability of an IgG1 fusion protein. IgG1 protein formulations were prepared by freeze-drying the protein with sucrose. Some samples were used as controls, and others were subjected to a further heat treatment (annealing). The protein structure was investigated with Fourier transform infrared spectroscopy (FTIR), and protein aggregation was monitored with size exclusion HPLC. Enthalpy recovery was studied using DSC, and global mobility represented by the structural relaxation time constant (tau(beta)) was characterized by a thermal activity monitor (TAM). The local mobility of the protein system was monitored by both (13)C solid-state NMR and neutron backscattering. Annealing increased the storage stability of the protein, as shown by the smaller aggregation rate and less total aggregation at the end of a storage period. The structural relaxation time constant of an annealed sample was significantly higher than the unannealed control sample, suggesting a decrease in global mobility of the protein system upon annealing. However, annealing does not significantly impact the protein secondary structure or the local mobility. Given the similar protein native structure and specific surface area, the improved stability upon annealing is mainly a result of reduced global molecular mobility.
本研究旨在探讨热处理对 IgG1 融合蛋白储存稳定性的影响。采用冷冻干燥法，用蔗糖制备 IgG1 蛋白制剂。部分样品作为对照，其余样品进行进一步的热处理（退火）。采用傅里叶变换红外光谱（FTIR）研究蛋白质结构，并采用尺寸排阻 HPLC 监测蛋白质聚集。通过差示扫描量热法（DSC）研究焓恢复，通过热活动监测仪（TAM）以结构弛豫时间常数（tau(beta)）表示整体流动性。通过 (13)C 固态 NMR 和背散射中子两种方法监测蛋白质体系的局部流动性。结果表明，退火可提高蛋白质的储存稳定性，表现为储存期末聚集率更小，总聚集量更少。退火样品的结构弛豫时间常数显著高于未经退火的对照样品，表明退火后蛋白质体系的整体流动性降低。然而，退火对蛋白质二级结构和局部流动性没有显著影响。鉴于相似的蛋白质天然结构和比表面积，退火后稳定性的提高主要是由于整体分子流动性降低所致。
