Li Yunsong, Williams Todd D, Topp Elizabeth M
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, 66047, USA.
Pharm Res. 2008 Feb;25(2):259-67. doi: 10.1007/s11095-007-9365-6. Epub 2007 Jun 28.
Excipients are added to lyophilized protein drug formulations to protect the protein during processing and storage, but the mechanisms are poorly understood. Here, hydrogen/deuterium (H/D) exchange with mass spectrometry was used to assess protein conformation and excipient interactions in lyophilized solids.
Calmodulin (CaM, 17 kD) was co-lyophilized with carbohydrate excipients (sucrose, mannitol, trehalose, raffinose, dextran 5,000, dextran 12,000) or guanidine hydrochloride (negative control) and exposed to D2O vapor at 33% RH and RT. Samples were then dissolved and analyzed by mass spectrometry (+ESI/MS). Peptic digestion provided additional, site-specific information on H/D exchange. Solids were further characterized by powder x-ray diffraction (PXRD), differential scanning calorimetry (DSC), infrared spectroscopy (FTIR) and water vapor sorption.
Excipients protected CaM from H/D exchange, increasing in the order guanidine hydrochloride < no excipient, mannitol < dextran 5,000, dextran 12,000 < sucrose < raffinose < trehalose. Effects were exerted primarily in the protein's alpha-helical segments.
The effects of carbohydrate excipients on protein conformation in lyophilized solids are not exhibited uniformly along the protein sequence, but instead are exerted in a site-specific manner. The results also demonstrate the utility of H/D exchange with ESI/MS for protein structure characterization in lyophilized samples.
向冻干蛋白质药物制剂中添加辅料以在加工和储存过程中保护蛋白质，但其作用机制尚不清楚。在此，采用氢/氘（H/D）交换质谱法评估冻干固体中的蛋白质构象和辅料相互作用。
将钙调蛋白（CaM，17 kD）与碳水化合物辅料（蔗糖、甘露醇、海藻糖、棉子糖、葡聚糖5000、葡聚糖12000）或盐酸胍（阴性对照）共冻干，并在33%相对湿度和室温下暴露于D2O蒸汽中。然后将样品溶解并用质谱法（+ESI/MS）进行分析。胃蛋白酶消化提供了有关H/D交换的额外的位点特异性信息。通过粉末X射线衍射（PXRD）、差示扫描量热法（DSC）、红外光谱（FTIR）和水蒸气吸附对固体进行进一步表征。
辅料保护CaM免受H/D交换，保护作用按以下顺序增强：盐酸胍＜无辅料、甘露醇＜葡聚糖5000、葡聚糖12000＜蔗糖＜棉子糖＜海藻糖。作用主要发生在蛋白质的α螺旋区段。
碳水化合物辅料对冻干固体中蛋白质构象的影响并非沿蛋白质序列均匀表现，而是以位点特异性方式发挥作用。结果还证明了H/D交换结合ESI/MS用于冻干样品中蛋白质结构表征的实用性。
