Winters M A, Knutson B L, Debenedetti P G, Sparks H G, Przybycien T M, Stevenson C L, Prestrelski S J
Department of Chemical Engineering, Princeton University, NJ 08544-5263, USA.
J Pharm Sci. 1996 Jun;85(6):586-94. doi: 10.1021/js950482q.
Supercritical CO2 was used as an antisolvent to form protein particles that exhibited minimal loss of activity upon reconstitution. Organic protein solutions were sprayed under a variety of operating conditions into the supercritical fluid, causing precipitation of dry, microparticulate (1-5 microns) protein powders. Three proteins were studied: trypsin, lysozyme, and insulin. Amide I band Raman spectra were used to estimate the alpha-helix and beta-sheet structural contents of native and precipitate powders of each protein. Analysis of the Raman spectral revealed minimal (lysozyme), intermediate (trypsin), and appreciable (insulin) changes in secondary structure with respect to the commercial starting materials. The perturbations in secondary structure suggest that the most significant event during supercritical fluid-induced precipitation involved the formation of beta-sheet structures with concomitant decreases of alpha-helix. Amide I band Raman and Fourier-transform infrared (FTIR) spectra indicate that higher operating temperatures and pressures lead to more extensive beta-sheet-mediated intermolecular interactions in the precipitates. Raman and FTIR spectra of redissolved precipitates are similar to those of aqueous commercial proteins, indicating that conformational changes were reversible upon reconstitution. These results suggest that protein precipitation in supercritical fluids can be used to form particles suitable for controlled release, direct aerosol delivery to the lungs, and long-term storage at ambient conditions.
超临界二氧化碳被用作抗溶剂来形成蛋白质颗粒，这些颗粒在重构后活性损失极小。在各种操作条件下，将有机蛋白质溶液喷入超临界流体中，导致干燥的微颗粒（1 - 5微米）蛋白质粉末沉淀。研究了三种蛋白质：胰蛋白酶、溶菌酶和胰岛素。利用酰胺I带拉曼光谱来估计每种蛋白质天然粉末和沉淀粉末中的α - 螺旋和β - 折叠结构含量。拉曼光谱分析表明，相对于市售起始原料，二级结构的变化最小（溶菌酶）、中等（胰蛋白酶）和明显（胰岛素）。二级结构的扰动表明，超临界流体诱导沉淀过程中最显著的事件涉及β - 折叠结构的形成以及α - 螺旋的相应减少。酰胺I带拉曼光谱和傅里叶变换红外（FTIR）光谱表明，较高的操作温度和压力会导致沉淀物中β - 折叠介导的分子间相互作用更为广泛。重新溶解的沉淀物的拉曼光谱和FTIR光谱与市售水性蛋白质的光谱相似，表明构象变化在重构后是可逆的。这些结果表明，超临界流体中的蛋白质沉淀可用于形成适合控释、直接气溶胶递送至肺部以及在环境条件下长期储存的颗粒。
