微流数字成像技术在单克隆抗体不溶性微粒检测中的应用

Application of Microflow Digital Imaging in Sub-visible Particle Detection of Monoclonal Antibodies

目的探讨微流数字成像技术(microflow digital imaging,MDI)在单克隆抗体(单抗)不溶性微粒检测中的应用价值。方法本实验首先采用聚苯乙烯乳胶微粒作为标准颗粒,研究了MDI方法的稳定性和准确性,之后采用MDI技术对两组单抗中≥10μm和≥25μm不溶性微粒的总数进行检测并对其微粒组成进行分析。一组为3家企业针对表皮生长因子受体(EGFR)靶点单抗,分析其不溶性微粒的组成与分布情况。另一组为某企业肿瘤坏死因子-α(TNF-α)靶点的单抗分别在4℃、37℃及6 000 lx光照条件下储存后的样品,观察不同储存条件对蛋白稳定性的影响。结果 MDI法对聚苯乙烯标准颗粒表现出较好的稳定性和准确性。通过对不溶性微粒的组成分析发现针对EGFR的单抗中,企业1和企业2不溶性微粒数量相当,但在非蛋白颗粒与蛋白颗粒占比上明显不同,企业3不溶性微粒数量远远大于企业1与企业2,且蛋白颗粒与非蛋白颗粒含量均较高,提示3家企业应采取不同策略控制不溶性微粒。通过对某企业TNF-α靶点的单抗在不同条件储存后的不溶性微粒进行分析发现,37℃及6 000 lx光照条件下储存后的样品蛋白颗粒数量急剧增加,说明储存条件对单抗中蛋白的稳定性有较大影响。结论 MDI技术不但能提供不溶性微粒粒径与数量的信息,还能提供形态和类别的信息,为不溶性微粒的控制以及单抗质量的提高提供依据。

OBJECTIVE To explore the application value of microflow digital imaging （MDI） in sub-visible particle detection of monoclonal antibodies（mAbs）. METHODS Polystyrene latex particles were adopted as standard particles to evaluate the stability and accuracy of MDI, then MDI was used to detect the quantities and compositions of sub-visible particles with diameters of ≥10μm and ≥25μm in two groups of mAbs. One group containing three mAbs from different companies against EGFR was selected to show the compositional difference of sub-visible particles. Another group was set to evaluate the impact of storage conditions on stability of sub-visible particles in mAb against TNF-α. The storage conditions included 4 ℃ , 37 ℃ and exposure under light of 6 000 Ix. RESULTS Using polystyrene latex particles, MDI method showed acceptable stability and accuracy. The analysis showed that the numbers of sub-visible particles in mAbs from Company 1 and Company 2 were comparable, but their main compositions were non-protein and protein particles separately. The number of sub-visible particles in mAb from Company 3 was much higher than that of Company 1 and Company 2 and the sub-visible particles included both non-protein and protein particles, indicating differed strategies should be adopted to control sub-visible particles. It was also found that the selected antibody against TNF-α was much more unstable when stored at 37 ℃ or exposed under light of 6 000 h as the quantities of protein particles were increased dramatically, which suggested the obvi- ous impact of storage conditions on stability of protein in mAbs. CONCLUSION MDI can provide not only diameters and the quantities of sub-visible particles, but also morphological information and tell the nature of the particles. These functions are powerful tools for sub-visible particle control and quality evaluation of mAbs.