Moreno M R, Tabitha T S, Nirmal J, Radhakrishnan K, Yee C H, Lim S, Venkatraman S, Agrawal R
School of Material Science & Engineering, Nanyang Technological University, Singapore.
School of Material Science & Engineering, Nanyang Technological University, Singapore.
Eur J Pharm Biopharm. 2016 Nov;108:156-167. doi: 10.1016/j.ejpb.2016.09.003. Epub 2016 Sep 8.
The anti-vascular endothelial growth factor (VEGF) agents such as ranibizumab (Lucentis®) and aflibercept (EyLea®) are currently used as monthly or bimonthly intravitreal injections to treat potentially retinal diseases such as wet age-related macular degeneration (AMD) or diabetic macular edema (DME). Because of the complications associated with repeated intra-vitreal injections, there is considerable interest in developing a sustained delivery system. The purpose of this study was to examine the stability of both therapeutic proteins under physiological conditions as well as when incorporated into drug delivery systems (DDS). First, thermotropic properties in physiological conditions and at different pH values were evaluated by differential scanning calorimetry (DSC) to determine the protein denaturation temperature. Second, the effects of pH and incubation time on conformational changes and aggregation were evaluated by circular dichroism (CD), steady-state tryptophan fluorescence spectroscopy, and size-exclusion chromatography (SEC). Also, the ability of both proteins to bind to VEGF was tested in the aforementioned experimental conditions for up to 30 days. Finally, we investigated the stability of both proteins after a rapid screening method that simulates the first homogenizing step during the protein microencapsulation process. This method allowed the development of stable ranibizumab and aflibercept formulations that may be useful to entrap these proteins into microparticles selecting the most convenient organic solvent and protein stabilizers.
抗血管内皮生长因子（VEGF）药物，如雷珠单抗（Lucentis®）和阿柏西普（EyLea®），目前用于每月或每两个月进行一次玻璃体内注射，以治疗潜在的视网膜疾病，如湿性年龄相关性黄斑变性（AMD）或糖尿病性黄斑水肿（DME）。由于反复玻璃体内注射会带来并发症，人们对开发一种持续给药系统有着浓厚的兴趣。本研究的目的是研究这两种治疗性蛋白质在生理条件下以及纳入药物递送系统（DDS）时的稳定性。首先，通过差示扫描量热法（DSC）评估生理条件下和不同pH值时的热致性质，以确定蛋白质变性温度。其次，通过圆二色性（CD）、稳态色氨酸荧光光谱和尺寸排阻色谱（SEC）评估pH和孵育时间对构象变化和聚集的影响。此外，在上述实验条件下测试这两种蛋白质与VEGF结合的能力，最长达30天。最后，我们在一种快速筛选方法后研究了这两种蛋白质的稳定性，该方法模拟了蛋白质微囊化过程中的第一步均质化步骤。这种方法有助于开发稳定的雷珠单抗和阿柏西普制剂，这些制剂可能有助于将这些蛋白质包裹入微粒中，同时选择最方便的有机溶剂和蛋白质稳定剂。
