Elkordy Amal A, Forbes Robert T, Barry Brian W
Drug Delivery Group, School of Pharmacy, University of Bradford, Bradford BD7 1DP, UK.
Int J Pharm. 2002 Oct 24;247(1-2):79-90. doi: 10.1016/s0378-5173(02)00379-4.
The development of proteins as therapeutic agents is challenging partly due to their inherent instabilities. Consequently, crystallisation and spray drying techniques were assessed to determine their effects on protein integrity using lysozyme as a model protein. Unprocessed, crystallised and spray-dried lysozyme were characterised by: thermal analysis using hot stage microscopy (HSM), differential scanning calorimetry (DSC), high sensitivity differential scanning calorimetry (HSDSC) and thermogravimetry (TGA); and spectroscopic analysis employing Fourier transform Raman (FT-Raman). Moisture contents were determined by TGA and Karl Fisher titration (KFT). Enzymatic assay measured biological activity. HSM showed no changes in crystals until complete melting. TGA and KFT indicated that spray-dried lysozyme contained a lower moisture content than crystals, hence the higher apparent thermal stability was shown by DSC. HSDSC revealed that crystallisation and spray drying did not affect the denaturation temperature of lysozyme in solution when compared with unprocessed material. However, in the solid state, FT-Raman spectra showed perturbation of the conformational structure of spray-dried sample, whereas crystal conformation remained intact. Enzymatic assay revealed increased activity retention of crystals compared with spray-dried powder. Hence, crystals maintained the conformational integrity and activity of lysozyme in solution.
蛋白质作为治疗剂的开发具有挑战性，部分原因在于其固有的不稳定性。因此，以溶菌酶作为模型蛋白，对结晶和喷雾干燥技术进行了评估，以确定它们对蛋白质完整性的影响。未处理的、结晶的和喷雾干燥的溶菌酶通过以下方法进行表征：使用热台显微镜（HSM）、差示扫描量热法（DSC）、高灵敏度差示扫描量热法（HSDSC）和热重分析法（TGA）进行热分析；以及采用傅里叶变换拉曼光谱（FT - Raman）进行光谱分析。通过TGA和卡尔费休滴定法（KFT）测定水分含量。酶活性测定测量生物活性。HSM显示在完全熔化之前晶体没有变化。TGA和KFT表明喷雾干燥的溶菌酶的水分含量低于晶体，因此DSC显示出更高的表观热稳定性。HSDSC表明，与未处理的材料相比，结晶和喷雾干燥不影响溶液中溶菌酶的变性温度。然而，在固态下，FT - Raman光谱显示喷雾干燥样品的构象结构受到扰动，而晶体构象保持完整。酶活性测定表明，与喷雾干燥粉末相比，晶体的活性保留增加。因此，晶体在溶液中保持了溶菌酶的构象完整性和活性。
