Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians-Universität München, Munich, Germany.
Department of Applied Science and Technology, Politecnico di Torino, Torino, Italy.
Eur J Pharm Biopharm. 2018 Nov;132:70-82. doi: 10.1016/j.ejpb.2018.09.004. Epub 2018 Sep 7.
Bulking agents as mannitol (Man) and glycine (Gly) require high bulking agent to stabilizer ratios to ensure their crystallization during the freeze-drying process. The aim of this study was to investigate several amino acids (AA) as potential alternative bulking agents in low AA to sucrose (Suc) ratios. A fast freeze-drying process was performed above the collapse temperature (Tc) of the amorphous phase challenging the crystalline AA scaffold. Lyophilizates and liquid formulations were characterized by differential scanning calorimetry. Karl-Fischer titration and X-ray powder diffraction as well as macroscopic cake appearance and reconstitution times were evaluated. Stability of a monoclonal antibody was investigated by UV-Vis spectroscopy, light obscuration and size exclusion chromatography. Phenylalanine (Phe), leucine (Leu) and isoleucine (Ile) crystallized upon freeze-drying at 5/45 and 10/40 AA/Suc ratios. 2.5/47.5 AA/Suc ratio showed less pronounced crystallization. Crystallization peaks were not suppressed by 2 or 50 mg/mL antibody in Leu and Ile lyophilizates. Reconstitution times could be improved by addition of surfactant. No signs of protein aggregation were detected after freeze-drying. Man and Gly yielded inacceptable cake appearance when dried with the fast freeze-drying cycle in such low bulking agent to Suc ratios. Leu, Ile and Phe can be used as alternate bulking agents at lower bulking agent to Suc ratios compared to Man or Gly. The selected AAs provided promising cake characteristics and good protein process stability.
作为赋形剂的甘露醇（Man）和甘氨酸（Gly）需要高赋形剂与稳定剂的比例，以确保它们在冷冻干燥过程中结晶。本研究的目的是研究几种氨基酸（AA）作为低 AA 与蔗糖（Suc）比例下潜在的替代赋形剂。在非晶相塌陷温度（Tc）以上进行快速冷冻干燥过程，对结晶 AA 支架构成挑战。通过差示扫描量热法、卡尔费休滴定和 X 射线粉末衍射以及宏观蛋糕外观和复溶时间对冻干赋形剂和液体配方进行了评估。通过紫外-可见光谱、光散射和尺寸排阻色谱法研究了单克隆抗体的稳定性。在 5/45 和 10/40 AA/Suc 比例下，苯丙氨酸（Phe）、亮氨酸（Leu）和异亮氨酸（Ile）在冷冻干燥时结晶。2.5/47.5 AA/Suc 比例显示出不太明显的结晶。在 Leu 和 Ile 的冻干赋形剂中，2 或 50mg/mL 抗体并未抑制结晶峰。添加表面活性剂可以改善复溶时间。冷冻干燥后未检测到蛋白质聚集的迹象。在如此低的赋形剂与 Suc 比例下，使用快速冷冻干燥循环干燥时，甘露醇和甘氨酸的蛋糕外观无法接受。与甘露醇或甘氨酸相比，Leu、Ile 和 Phe 可作为替代赋形剂，用于较低的赋形剂与 Suc 比例。所选 AA 提供了有前景的蛋糕特性和良好的蛋白质工艺稳定性。
