Grup d'Enginyeria Cellular i Bioprocessos, Departament d'Enginyeria Química, Biològica i Ambiental, Escola d'Enginyeria, Universitat Autònoma de Barcelona, Barcelona, Spain.
Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands.
Biotechnol Bioeng. 2022 May;119(5):1207-1221. doi: 10.1002/bit.28051. Epub 2022 Feb 10.
Human immunodeficiency virus 1 (HIV-1) virus-like particles (VLPs) are nanostructures derived from the self-assembly and cell budding of Gag polyprotein. Mimicking the native structure of the virus and being noninfectious, they represent promising candidates for the development of new vaccines as they elicit a strong immune response. In addition to this, the bounding membrane can be functionalized with exogenous antigens to target different diseases. Protein glycosylation depends strictly on the production platform and expression system used and the displayed glycosylation patterns may influence downstream processing as well as the immune response. One of the main challenges for the development of Gag VLP production bioprocess is the separation of VLPs and coproduced extracellular vesicles (EVs). In this study, porous graphitized carbon separation method coupled with mass spectrometry was used to characterize the N- and O- glycosylation profiles of Gag VLPs produced in HEK293 cells. We identified differential glycan signatures between VLPs and EVs that could pave the way for further separation and purification strategies to optimize downstream processing and move forward in VLP-based vaccine production technology.
人类免疫缺陷病毒 1（HIV-1）病毒样颗粒（VLPs）是源自 Gag 多蛋白的自我组装和细胞出芽的纳米结构。它们模拟病毒的天然结构且无感染性，作为新型疫苗的候选物具有很大的潜力，因为它们能引发强烈的免疫反应。此外，包膜可以通过外源性抗原进行功能化，以针对不同的疾病。蛋白质糖基化严格依赖于所使用的生产平台和表达系统，并且所展示的糖基化模式可能会影响下游处理以及免疫反应。Gag VLP 生产生物工艺开发的主要挑战之一是 VLPs 和共产生的细胞外囊泡（EVs）的分离。在这项研究中，使用多孔石墨化碳分离方法结合质谱法来表征在 HEK293 细胞中产生的 Gag VLPs 的 N-和 O-糖基化谱。我们确定了 VLPs 和 EVs 之间的差异聚糖特征，这可能为进一步的分离和纯化策略铺平道路，以优化下游处理并推动基于 VLP 的疫苗生产技术的发展。
