González-Domínguez Irene, Lorenzo Elianet, Bernier Alice, Cervera Laura, Gòdia Francesc, Kamen Amine
Departament d'Enginyeria Química Biològica i Ambiental, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain.
Department of Bioengineering, McGill University, Montreal, QC H3A 0E9, Canada.
Vaccines (Basel). 2021 Oct 9;9(10):1154. doi: 10.3390/vaccines9101154.
Gag-based virus-like particles (VLPs) have high potential as scaffolds for the development of chimeric vaccines and delivery strategies. The production of purified preparations that can be preserved independently from cold chains is highly desirable to facilitate distribution and access worldwide. In this work, a nimble purification has been developed, facilitating the production of Gag VLPs. Suspension-adapted HEK 293 cells cultured in chemically defined cell culture media were used to produce the VLPs. A four-step downstream process (DSP) consisting of membrane filtration, ion-exchange chromatography, polishing, and lyophilization was developed. The purification of VLPs from other contaminants such as host cell proteins (HCP), double-stranded DNA, or extracellular vesicles (EVs) was confirmed after their DSP. A concentration of 2.2 ± 0.8 × 10 VLPs/mL in the lyophilized samples was obtained after its storage at room temperature for two months. Morphology and structural integrity of purified VLPs was assessed by cryo-TEM and NTA. Likewise, the purification methodologies proposed here could be easily scaled up and applied to purify similar enveloped viruses and vesicles.
基于Gag的病毒样颗粒（VLPs）作为嵌合疫苗和递送策略开发的支架具有很高的潜力。非常希望生产出能够独立于冷链保存的纯化制剂，以促进全球范围内的分发和获取。在这项工作中，开发了一种灵活的纯化方法，以促进Gag VLPs的生产。使用在化学成分确定的细胞培养基中培养的悬浮适应型HEK 293细胞来生产VLPs。开发了一个由膜过滤、离子交换色谱、精制和冻干组成的四步下游工艺（DSP）。在其DSP之后，证实了从其他污染物如宿主细胞蛋白（HCP）、双链DNA或细胞外囊泡（EVs）中纯化VLPs。冻干样品在室温下储存两个月后，获得的浓度为2.2±0.8×10 VLPs/mL。通过冷冻电镜和纳米颗粒跟踪分析（NTA）评估纯化VLPs的形态和结构完整性。同样，这里提出的纯化方法可以很容易地扩大规模，并应用于纯化类似的包膜病毒和囊泡。
