Chang Liuquan Lucy, Shepherd Deanna, Sun Joanna, Ouellette David, Grant Kathleen L, Tang Xiaolin Charlie, Pikal Michael J
Department of Pharmaceutical Science, School of Pharmacy, University of Connecticut, Storrs, CN 06269, USA.
J Pharm Sci. 2005 Jul;94(7):1427-44. doi: 10.1002/jps.20364.
The purpose of this study is to investigate the mechanism of protein stabilization by sugars in the solid state. That is, explore whether the stabilization is controlled by "glass dynamics" or by native structure preservation through "specific interaction" between sugars and protein. The IgG1 antibody (150 kD) and recombinant human serum albumin (rHSA) (65 kD) were formulated with sorbitol, trehalose, and sucrose. Degradation of lyophilized formulations was quantified using size exclusion (SEC) and ion-exchange chromatography (IEX). The secondary structure of the protein in these formulations was characterized using Fourier Transform Infrared (FTIR) spectroscopy. The molecular mobility, as measured by the stretched relaxation time (tau(beta)) was obtained by fitting the modified stretched exponential (MSE) equation to the calorimetric data from the Thermal Activity Monitor (TAM). Compared with sucrose and trehalose, sorbitol could only slightly protect the protein against aggregation and had no effect on chemical degradation. The chemical degradation and aggregation rates of the protein decreased when the weight ratio of sucrose to protein increased from 0 to 2:1. Storage stability of the proteins showed a reasonably good correlation with the degree of retention of native structure of protein during drying as measured by the spectral correlation coefficient for FTIR spectra. The plots of tau(beta) as a function of fraction of sucrose passed through a maximum at 1:1 weight ratio of sucrose to protein. That is, the molecular mobility did not correlate with the stability of protein at high levels of sucrose content. Although the glass transition appears to be an important parameter for stability, protein stabilization by sugars in the solid state cannot be wholly explained by the glass dynamics mechanism, at least as measured by tau(beta). However, it is possible that the beta-relaxations rather than the alpha-relaxations (i.e., the tau we measured) are critical to stability. The data show that storage stability correlates best with "structure" as determined by FTIR spectroscopy. However, while a specific interaction between stabilizer and protein might be responsible for the preservation of native structure, the evidence supporting this position is not compelling.
本研究的目的是探究糖类在固态下对蛋白质的稳定机制。也就是说，探讨这种稳定性是由“玻璃态动力学”控制，还是通过糖类与蛋白质之间的“特异性相互作用”来保持天然结构。用山梨醇、海藻糖和蔗糖配制了IgG1抗体（150 kD）和重组人血清白蛋白（rHSA）（65 kD）。使用尺寸排阻色谱（SEC）和离子交换色谱（IEX）对冻干制剂的降解进行定量。用傅里叶变换红外（FTIR）光谱对这些制剂中蛋白质的二级结构进行表征。通过将修正的伸展指数（MSE）方程拟合到热活性监测仪（TAM）的量热数据中，获得由伸展弛豫时间（tau(beta)）测量的分子流动性。与蔗糖和海藻糖相比，山梨醇只能略微保护蛋白质不发生聚集，且对化学降解没有影响。当蔗糖与蛋白质的重量比从0增加到2:1时，蛋白质的化学降解和聚集速率降低。蛋白质的储存稳定性与干燥过程中蛋白质天然结构的保留程度呈现出合理的良好相关性，该保留程度通过FTIR光谱的光谱相关系数来衡量。tau(beta)作为蔗糖分数的函数图在蔗糖与蛋白质重量比为1:1时通过最大值。也就是说，在高蔗糖含量水平下，分子流动性与蛋白质的稳定性不相关。尽管玻璃化转变似乎是稳定性的一个重要参数，但糖类在固态下对蛋白质的稳定作用不能完全用玻璃态动力学机制来解释，至少通过tau(beta)测量是这样。然而，有可能β弛豫而非α弛豫（即我们测量的tau）对稳定性至关重要。数据表明，储存稳定性与FTIR光谱测定的“结构”相关性最佳。然而，虽然稳定剂与蛋白质之间的特异性相互作用可能是天然结构得以保留的原因，但支持这一观点的证据并不确凿。
