Pikal M J, Rigsbee D R
Lilly Research Laboratories, Eli Lilly & Co., Indianapolis, Indiana 46285, USA.
Pharm Res. 1997 Oct;14(10):1379-87. doi: 10.1023/a:1012164520429.
Generalizations based upon behavior of small molecules have established that a crystalline solid is generally much more stable toward chemical degradation than is the amorphous solid. This study examines the validity of this generalization for proteins using biosynthetic human insulin as the model protein.
Amorphous insulin was prepared by freeze drying the supernate from a suspension of zinc insulin crystals adjusted to pH 7.1. Storage stability at 25 degrees C and 40 degrees C were compared for the freeze dried material, the dried suspended crystals, and the starting batch of crystals. Samples were equilibrated at selected relative humidities between zero and 75% to obtain samples at various water contents. Assays for dimer formation were performed by size exclusion HPLC and assays for deamidated product were carried out by reverse phase HPLC. Degradation was found to be linear in square root of time, and the slopes from % degradation vs. square root of time were used to define the rate constants for degradation. Differential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopy (FTIR) were used to characterize the state of the protein in the solids.
As expected based upon previous results, the primary degradation pathways involve deamidation at the AsnA21 site and co-valent dimer formation, presumably involving the A-21 site. Contrary to expectations, amorphous insulin is far more stable than crystalline insulin under all conditions investigated. While increasing water content increases the rate of degradation of crystalline insulin, rate constants for degradation in the amorphous solid are essentially independent of water content up to the maximum water content studied (approximately 15%).
Based upon the FTIR and DSC data, both crystalline and amorphous insulin retain some higher order structure when dried, but the secondary structure is significantly perturbed from that characteristic of the native solution state. However, neither DSC nor FTIR data provide a clear interpretation of the difference in stability between the amorphous and crystalline solids. The mechanism responsible for the superior stability of amorphous insulin remains obscure.
基于小分子行为的归纳表明，结晶固体通常比无定形固体对化学降解更稳定。本研究以生物合成人胰岛素作为模型蛋白，检验这一归纳对蛋白质的有效性。
通过冷冻干燥将锌胰岛素晶体悬浮液调至pH 7.1后的上清液制备无定形胰岛素。比较了冷冻干燥材料、干燥的悬浮晶体和起始晶体批次在25℃和40℃下的储存稳定性。将样品在0至75%的选定相对湿度下平衡，以获得不同含水量的样品。通过尺寸排阻高效液相色谱法进行二聚体形成的测定，通过反相高效液相色谱法进行脱酰胺产物的测定。发现降解与时间的平方根呈线性关系，降解百分比与时间平方根的斜率用于定义降解速率常数。差示扫描量热法（DSC）和傅里叶变换红外光谱法（FTIR）用于表征固体中蛋白质的状态。
基于先前结果预期，主要降解途径包括AsnA21位点的脱酰胺和共价二聚体形成，推测涉及A - 21位点。与预期相反，在所有研究条件下，无定形胰岛素比结晶胰岛素稳定得多。虽然含水量增加会提高结晶胰岛素的降解速率，但在无定形固体中，直至所研究的最大含水量（约15%），降解速率常数基本与含水量无关。
基于FTIR和DSC数据，结晶胰岛素和无定形胰岛素干燥后均保留一些高级结构，但二级结构与天然溶液状态的特征有显著差异。然而，DSC和FTIR数据均未对无定形和结晶固体稳定性差异提供清晰解释。无定形胰岛素稳定性优越的机制仍不清楚。
