Sukumar Muppalla, Storms Sacha M, De Felippis Michael R
Biopharmaceutical Research and Development, Eli Lilly and Company, Indianapolis, Indiana, USA.
Pharm Res. 2005 May;22(5):789-96. doi: 10.1007/s11095-005-2596-5. Epub 2005 May 17.
Manufacturing processes expose protein pharmaceuticals to organic solvents that may perturb the native folded state, increasing the potential for irreversible aggregation or surface adsorption. The aim of this study was to characterize the conformational states of human growth hormone (hGH) in aqueous ethanolic solutions.
The higher order structure of hGH was investigated using far- and near-UV circular dichroism (CD) and fluorescence spectroscopy as orthogonal techniques, and the hydrodynamic size was monitored using dynamic light scattering.
CD data suggested that the secondary structure of hGH remained unchanged up to 50% (v/v) ethanol, but the tertiary structure was perturbed at a20% ethanol. Fluorescence anisotropy, however, showed that the mobility of the buried Trp residue was restricted even at 30% ethanol, suggesting a differently packed structural core in 30% ethanol relative to the native structure. Consistent with this result, thermal unfolding of hGH in 30% ethanol was more facile compared to that in 0% and 20% ethanol. At >40% ethanol, fluorescence data were consistent with increased solvent exposure of the tryptophan.
The results point to progressive unfolding of hGH that increases solvent exposure of the hydrophobic core as a function of ethanol concentration and suggest that non-native intermediate states are populated in 30-60% ethanol.
制造工艺会使蛋白质药物暴露于有机溶剂中，这些有机溶剂可能会扰乱天然折叠状态，增加不可逆聚集或表面吸附的可能性。本研究的目的是表征人生长激素（hGH）在乙醇水溶液中的构象状态。
使用远紫外和近紫外圆二色性（CD）以及荧光光谱作为正交技术研究hGH的高级结构，并使用动态光散射监测流体动力学尺寸。
CD数据表明，在乙醇体积分数高达50%时，hGH的二级结构保持不变，但在乙醇体积分数为20%时，三级结构受到扰动。然而，荧光各向异性表明，即使在乙醇体积分数为30%时，埋藏的色氨酸残基的流动性也受到限制，这表明相对于天然结构，在乙醇体积分数为30%时结构核心的堆积方式不同。与该结果一致，与在乙醇体积分数为0%和20%时相比，hGH在乙醇体积分数为30%时的热解折叠更容易。在乙醇体积分数>40%时，荧光数据与色氨酸暴露于溶剂的增加一致。
结果表明hGH会随着乙醇浓度的增加而逐渐解折叠，导致疏水核心暴露于溶剂中，并表明在乙醇体积分数为30%-60%时会形成非天然中间态。
