Winters M A, Debenedetti P G, Carey J, Sparks H G, Sane S U, Przybycien T M
Department of Chemical Engineering, Princeton University, New Jersey 08544-5263, USA.
Pharm Res. 1997 Oct;14(10):1370-8. doi: 10.1023/a:1012112503590.
The long-term and high-temperature storage of dry, micron-sized particles of lysozyme, trypsin, and insulin was investigated. Subsequent to using supercritical carbon dioxide as an antisolvent to induce their precipitation from a dimethylsulfoxide solution, protein microparticles were stored in sealed containers at -25, -15, 0, 3, 20, 22, and 60 degrees C. The purpose of this study was to investigate the suitability of supercritical antisolvent precipitation as a finishing step in protein processing.
Karl Fisher titrations were used to determine the residual moisture content of commercial and supercritically-processed protein powders. The secondary structure of the dry protein particles was determined periodically during storage using Raman spectroscopy. The proteins were also redissolved periodically in aqueous buffers and assayed spectrophotometrically for biological activity and by circular dichroism for structural conformation in solution.
Amide I band Raman spectra indicate that the secondary structure of the protein particles, while perturbed from that of the solution state, remained constant in time, regardless of the storage temperature. The recoverable biological activity upon reconstitution for the supercritically-processed lysozyme and trypsin microparticles was also preserved and found to be independent of storage temperature. Far UV circular dichroism spectra support the bioactivity assays and further suggest that adverse structural changes, with potential to hinder renaturation upon redissolution, do not take place during storage.
The present study suggests that protein precipitation using supercritical fluids may yield particles suitable for long-term storage at ambient conditions.
研究了溶菌酶、胰蛋白酶和胰岛素的干燥微米级颗粒的长期高温储存情况。在使用超临界二氧化碳作为抗溶剂诱导它们从二甲基亚砜溶液中沉淀后，将蛋白质微粒储存在密封容器中，温度分别为-25、-15、0、3、20、22和60摄氏度。本研究的目的是探讨超临界抗溶剂沉淀作为蛋白质加工最后一步的适用性。
使用卡尔费休滴定法测定市售和超临界处理的蛋白质粉末的残留水分含量。在储存期间定期使用拉曼光谱法测定干燥蛋白质颗粒的二级结构。还定期将蛋白质重新溶解在水性缓冲液中，通过分光光度法测定其生物活性，并通过圆二色性测定溶液中的结构构象。
酰胺I带拉曼光谱表明，蛋白质颗粒的二级结构虽然与溶液状态有所不同，但随时间保持不变，与储存温度无关。超临界处理的溶菌酶和胰蛋白酶微粒重新溶解后的可恢复生物活性也得以保留，且发现与储存温度无关。远紫外圆二色光谱支持生物活性测定，并进一步表明在储存过程中不会发生可能阻碍重新溶解时复性的不利结构变化。
本研究表明，使用超临界流体沉淀蛋白质可能产生适合在环境条件下长期储存的颗粒。
