退火对冻干融合蛋白依那西普产品稳定性及可见气泡的影响

Effects of Annealing on Product Stability and Visible Bubbles of The Freeze-dried Fusion Protein Etanercept Formulations

关键词:  退火  气泡  依那西普  冷冻干燥  复溶  稳定性

Key words:  annealing, bubble, etanercept, freeze-drying, reconstitution, stability

摘要:

目的 本研究旨在研究冻干过程中的退火步骤对冻干依那西普产品复溶过程中可见气泡和其稳定性的影响。 方法 本研究采用不同退火温度冻干得到依那西普产品，通过目视观察冻干粉外观，卡尔费休法测冻干粉水分，游标卡尺测量冻干粉复溶后的气泡体积，扫描电镜观察冻干粉体的内部微观结构，X射线衍射检测辅料结晶情况，确定退火对冻干粉末的物理性质的影响。随后进行加速稳定性实验，将冻干制剂分别放置于40 ℃储存1、2、3个月，用分子排阻色谱法考察不同时期的可溶性聚体含量，傅里叶变换红外光谱观察零时和3个月后冻干制剂的稳定性情况。 结果 实验结果显示退火可以减少冻干依那西普产品复溶后可见气泡的体积，不同退火温度减少的效果接近；扫描电镜结果显示退火会增大冻干粉微孔体积，进而在复溶时产生更大气泡；X射线衍射结果显示退火与否不影响辅料甘露醇结晶晶型；在加速稳定性实验中，分子排阻色谱法结果显示退火会略微降低冻干依那西普制剂的稳定性，不同退火温度降低的效果接近；红外光谱结果显示各组光谱中的曲线在特征峰上重叠，说明在加速实验中依那西普的二级结构基本保持不变。 结论 退火可以减少复溶后的可见气泡体积，并略微降低依那西普制剂的稳定性，在冻干时设置退火步骤可以有效减少复溶后的气泡问题。

Abstract:

ABSTRACT: OBJECTIVE The aim of this study was to investigate the effect of annealing steps during freeze-drying on the visible bubbles and their stability during the reconstitution of freeze-drying etanercept products. METHODS In this study, etanercept products were obtained by freeze-drying at different annealing temperatures. The appearance of the freeze-dried powder was observed visually, the moisture content of the freeze-dried powder was measured by Carl Fischer method, the bubble volume after reconstitution was measured by vernier caliper, the internal microstructure of the freeze-dried powder was observed by scanning electron microscopy, and the crystallization of the excipients was detected by X-ray diffraction, and accelerated stability experiments were conducted by storing at 40 ℃ for 1, 2, and 3 months respectively. Then the soluble aggregates in different periods was investigated by size exclusion chromatography, and the stability of freeze-dried formulations before and after 3 months storage was observed by Fourier transform infrared spectroscopy. RESULTS The experimental results show that annealing can reduce the volume of visible bubbles after reconstitution of freeze-dried etanercept products, and the reduction effect is similar with different annealing temperatures. SEM results showed that annealing increased the size of freeze-dried powder micropores, which resulted in larger bubbles during reconstitution. X-ray diffraction results show that annealing does not affect the crystal type of mannitol. In the accelerated stability test, the results of size exclusion chromatography show that annealing can slightly reduce the stability of freeze-dried etanercept formulation, and the effect of different annealing temperatures is similar. The infrared spectra showed that the curves in each group overlapped on the characteristic peaks, indicating that the secondary structure of etanercept remained basically unchanged in the accelerated experiment. CONCLUSION Annealing can reduce the visible bubble volume after reconstitution and slightly reduce the stability of etanercept formulations, and setting the annealing step during lyophilization can effectively reduce the bubble problem after reconstitution.