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首页> 外文期刊>Vaccine >Process intensification for high yield production of influenza H1N1 Gag virus-like particles using an inducible HEK-293 stable cell line
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Process intensification for high yield production of influenza H1N1 Gag virus-like particles using an inducible HEK-293 stable cell line

机译：使用诱导HEK-293稳定细胞系加入高产型流感H1N1 GAG病毒样颗粒的加强

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Influenza virus dominant antigens presentation using virus like particle (VLP) approach is attractive for the development of new generation of influenza vaccines. Mammalian cell platform offers many advantages for VLP production. However, limited attention has been paid to the processing of mammalian cell produced VLPs. Better understanding of the production system could contribute to increasing the yields and making large-scale VLP vaccine manufacturing feasible. In a previous study, we have generated a human embryonic kidney HEK-293 inducible cell line expressing Hemagglutinin (HA) and Neuraminidase (NA), which was used to produce VLPs upon transient transfection with a plasmid containing HIV-1 Gag. In this work, to streamline the production process, we have developed a new HEK-293 inducible cell line adapted to suspension growth expressing the three proteins HA, NA (H1N1 A/PR/8/1934) and the Gag fused to GFP for monitoring the VLP production. The process was optimized to reach higher volumetric yield of VLPs by increasing the cell density at the time of induction without sacrificing the cell specific productivity. A 5-fold improvement was achieved by doing media evaluation at small scale. Furthermore, a 3-L perfusion bioreactor mirrored the performance of small-scale shake flask cultures with sequential medium replacement. The cell density was increased to 14 x 10(6) cells/ml at the time of induction which augmented by 60-fold the volumetric yield to 1.54 x 10(10) Gag-GFP fluorescent events/ml, as measured by flow cytometry. The 9.5-L harvest from the perfusion bioreactor was concentrated by tangential flow filtration at low shear rate. The electron micrographs revealed the presence of VLPs of 100-150 nm with the characteristic dense core of HIV-1 particles. The developed process shows the feasibility of producing high quantity of influenza VLPs from an inducible mammalian stable cell line aiming at large scale vaccine manufacturing. Crown Copyright (C) 2017 Published by Elsevier Ltd. All rights reserved.
机译：流感病毒的致抗原呈现使用粒子（VLP）方法的病毒是有吸引力的，对于新一代流感疫苗具有吸引力。哺乳动物细胞平台为VLP生产提供了许多优势。然而，已经有限地注意哺乳动物细胞产生的VLP。更好地了解生产系统可能有助于提高产量和制造大规模的VLP疫苗制造可行。在先前的研究中，我们已经产生了表达血血糖素（HA）和神经氨酸酶（NA）的人胚胎肾HEK-293诱导细胞系，其用于在瞬时转染时用含有HIV-1 GAG的质粒产生VLP。在这项工作中，为了简化生产过程，我们开发了一种新的HEK-293诱导细胞系，适用于表达三种蛋白质HA，NA（H1N1 A / PR / 1934）的悬浮生长，并融合给GFP进行监测的GAG VLP生产。优化该方法以通过增加诱导时的细胞密度而不牺牲细胞特异性生产率来达到较高的VLP的体积产率。通过小规模进行媒体评估实现了5倍的改善。此外，3L灌注生物反应器反应着具有顺序介质的小尺寸摇瓶培养物的性能。在诱导时，细胞密度将细胞密度增加到14×10（6）个细胞/ mL，其通过流式细胞术测量，增加了60倍的体积率至1.54×10（10）个GG-GFP荧光事件/ mL。通过以低剪切速率的切向流过滤浓缩来自灌注生物反应器的9.5 -L。电子显微照片揭示了100-150nm的VLP，具有HIV-1颗粒的特征致密核心。开发的方法表明，从旨在大规模疫苗制造的诱导哺乳动物稳定的细胞系中产生大量流感VLP的可行性。 Crown版权所有（c）2017由elestvier有限公司出版。保留所有权利。

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