Advancements in the field of characterization techniques have broadened the opportunities to deepen into nanoparticle production bioprocesses. Gag‐based virus‐like particles (VLPs) have shown their potential as candidates for recombinant vaccine development. However, comprehensive characterization of the production process is still a requirement to meet the desired critical quality attributes. In this work, the production process of Gag VLPs by baculovirus (BV) infection in the reference High Five and Sf9 insect cell lines is characterized in detail. To this end, the Gag polyprotein was fused in frame to the enhanced green fluorescent protein (eGFP) to favor process evaluation with multiple analytical tools. Tracking of the infection process using confocal microscopy and flow cytometry revealed a pronounced increase in the complexity of High Five over Sf9 cells. Cryogenic transmission electron microscopy (cryo‐TEM) characterization determined that changes in cell complexity could be attributed to the presence of occlusion‐derived BV in High Five cells, whereas Sf9 cells evidenced a larger proportion of the budded virus phenotype (23‐fold). Initial evaluation of the VLP production process using spectrofluorometry showed that higher levels of the Gag‐eGFP polyprotein were obtained in High Five cells (3.6‐fold). However, comparative analysis based on nanoparticle quantification by flow virometry and nanoparticle tracking analysis (NTA) proved that Sf9 cells were 1.7‐ and 1.5‐fold more productive in terms of assembled VLPs, respectively. Finally, analytical ultracentrifugation coupled to flow virometry evidenced a larger sedimentation coefficient of High Five‐derived VLPs, indicating a possible interaction with other cellular compounds. Taken together, these results highlight the combined use of microscopy and flow cytometry techniques to improve vaccine development processes using the insect cell/BV expression vector system. © 2020 International Society for Advancement of Cytometry

中文翻译：

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耦合显微镜和流式细胞术用于昆虫细胞中纳米颗粒生产的综合表征。

表征技术领域的进步拓宽了深入研究纳米颗粒生产生物过程的机会。基于 Gag 的病毒样颗粒 (VLP) 已显示出其作为重组疫苗开发候选者的潜力。然而，生产过程的综合表征仍然是满足所需的关键质量属性的要求。在这项工作中，详细描述了参考 High Five 和 Sf9 昆虫细胞系中杆状病毒 (BV) 感染 Gag VLP 的生产过程。为此，将 Gag 多聚蛋白与增强型绿色荧光蛋白 (eGFP) 框内融合，以支持使用多种分析工具进行过程评估。使用共聚焦显微镜和流式细胞术跟踪感染过程显示 High Five 比 Sf9 细胞的复杂性显着增加。低温透射电子显微镜 (cryo-TEM) 表征确定细胞复杂性的变化可归因于 High Five 细胞中存在闭塞衍生的 BV，而 Sf9 细胞证明了更大比例的出芽病毒表型（23 倍）。使用荧光分光光度计对 VLP 生产过程的初步评估表明，在 High Five 细胞中获得了更高水平的 Gag-eGFP 多蛋白（3.6 倍）。然而，基于通过流动病毒测定法和纳米粒子追踪分析 (NTA) 进行的纳米粒子量化的比较分析证明，Sf9 细胞在组装 VLP 方面的生产力分别提高了 1.7 倍和 1.5 倍。最后，分析超速离心与流动病毒测定相结合证明了 High Five 衍生的 VLP 的沉降系数更大，表明可能与其他细胞化合物相互作用。总之，这些结果突出了显微镜和流式细胞术技术的结合使用，以使用昆虫细胞/BV 表达载体系统改进疫苗开发过程。© 2020 国际细胞计量学促进会