基因治疗产品中病毒颗粒的微粒特性研究

目的： 研究基因治疗产品中病毒颗粒的微粒特性及粒径分布情况。方法： 采用扫描电镜、动态光散射法、微流数字成像法、光阻法及目测法对腺病毒（adenovirus，AdV）、腺相关病毒（adeno-associated virus，AAV）和单纯疱疹病毒（herpes simplex virus，HSV）基因治疗产品中病毒颗粒的微粒特性、粒径分布及制剂外观进行观察研究。结果： 扫描电镜结果显示，AdV、AAV、HSV均存在不完整颗粒（空心病毒颗粒、衣壳碎片），HSV病毒存在包膜脱落或融合的情况；动态光散射法结果显示AdV对温度敏感，易形成聚集体，AAV的粒径分布不均一，变异度大，HSV实测粒径偏大且分布不均一；微流数字成像法、光阻法及目测法结果提示样品中存在的50 μm以上的微粒，与典型的蛋白聚集体形态相似，且50 μm以上的颗粒数与液体的浊度呈正相关，100 μm以上颗粒与絮状沉淀物相关。结论： 不同病毒的微粒特性也不相同，微粒分布情况及动态变化过程与聚集体的形成有关，可采用多种方法优势互补，对微粒进行全范围监测。

Objective: To study the particle characteristics and size distribution of virion in gene therapy products. Methods: The particle characteristics,size distribution and product appearance of adenovirus (AdV),adeno-associated virus(AAV) and herpes simplex virus (HSV) were observed and studied by transmission electron microscope (TEM),dynamic light scattering (DLS),microflow digital imaging (MDI),light obscuration and visual observation.Results: The results of TEM indicated that incomplete virus particles (empty particles and fragments) existed in AdV,AAV and HSV samples,and "takeoff" or "fusion" of the envelope were found in HSV samples;the results of DLS demonstrated that AdV was sensitive to temperature and tended to form aggregates,particle size distribution of AAV was inhomogeneous and variable;the results of MDI,light obscuration and visual observation suggested that the morphology of particles above 50 μm existed in samples was similar to typical protein aggregates. The number of particles above 50 μm was positively related to the turbidity of solutions,and the number of particles above 100 μm was related to the precipitate.Conclusion: The particle characteristics of different virus differ from each other,and the distribution of particles and its dynamic change process are related to the formation of aggregates.Full-range monitoring of particles can be achieved by a series of complementary methods.

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