Heller M C, Carpenter J F, Randolph T W
Department of Chemical Engineering, University of Colorado at Boulder, Boulder, Colorado 80309-0424, USA.
J Pharm Sci. 1999 Jan;88(1):58-64. doi: 10.1021/js980257j.
PEGylation of proteins is of great interest to the pharmaceutical industry as covalent attachment of poly(ethylene glycol) (PEG) molecules can increase protein sera half-lives and reduce antigenicity. Not surprisingly, PEGylation significantly alters the surface characteristics of a protein, and consequently, its conformational stability during freezing and drying. Freeze concentration-induced phase separation between excipients has been previously shown to cause degradation of the secondary structure in lyophilized hemoglobin. In this report we show how PEGylation of two proteins, hemoglobin- and brain-derived neurotrophic factor (BDNF), influences partitioning and protein secondary structure as determined by FTIR spectroscopy in a system prone to freezing-induced phase separation. PEGylation of hemoglobin reduces the loss of structure induced by lyophilization in a PEG/dextran system that phase separates during freezing, perhaps due to altered partitioning. The partition coefficient for native hemoglobin favors the dextran-rich phase (PEG/dextran partition coefficient = 0.3), while PEGylated hemoglobin favors the PEG phase (partition coefficient = 3.1). In addition, we demonstrate that PEGylation alters hemoglobin's stability during lyophilization in the absence of other excipients. In contrast, because native BDNF already partitions into the PEG-rich phase, PEGylation of BDNF has a less dramatic effect on both partition coefficients and conformational stability during lyophilization. This is the first report on the effects of PEGylation on protein structural stability during lyophilization and points out the need to consider modification of formulations in response to changing protein surface characteristics.
蛋白质的聚乙二醇化对制药行业具有重大意义，因为聚（乙二醇）（PEG）分子的共价连接可以延长蛋白质在血清中的半衰期并降低抗原性。不出所料，聚乙二醇化显著改变了蛋白质的表面特性，因此也改变了其在冷冻和干燥过程中的构象稳定性。先前已经表明，冻干赋形剂之间的冷冻浓缩诱导相分离会导致冻干血红蛋白二级结构的降解。在本报告中，我们展示了两种蛋白质——血红蛋白和脑源性神经营养因子（BDNF）的聚乙二醇化如何影响在易于发生冷冻诱导相分离的系统中通过傅里叶变换红外光谱（FTIR）测定的分配和蛋白质二级结构。血红蛋白的聚乙二醇化减少了在冷冻过程中发生相分离的PEG/右旋糖酐系统中冻干诱导的结构损失，这可能是由于分配的改变。天然血红蛋白的分配系数有利于富含右旋糖酐的相（PEG/右旋糖酐分配系数 = 0.3），而聚乙二醇化血红蛋白则有利于PEG相（分配系数 = 3.1）。此外，我们证明在没有其他赋形剂的情况下，聚乙二醇化会改变血红蛋白在冻干过程中的稳定性。相比之下，由于天然BDNF已经分配到富含PEG的相中，BDNF的聚乙二醇化对冻干过程中的分配系数和构象稳定性的影响较小。这是关于聚乙二醇化对冻干过程中蛋白质结构稳定性影响的首次报告，并指出需要根据蛋白质表面特性的变化考虑调整配方。
