Correia Ricardo, Fernandes Bárbara, Alves Paula M, Carrondo Manuel J T, Roldão António
IBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal.
ITQB NOVA, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.
Vaccines (Basel). 2020 Oct 7;8(4):589. doi: 10.3390/vaccines8040589.
The use of non-standard culture conditions has proven efficient to increase cell performance and recombinant protein production in different cell hosts. However, the establishment of high-producing cell populations through adaptive laboratory evolution (ALE) has been poorly explored, in particular for insect cells. In this study, insect High Five cells were successfully adapted to grow at a neutral culture pH (7.0) through ALE for an improved production of influenza hemagglutinin (HA)-displaying virus-like particles (VLPs). A stepwise approach was used for the adaptation process, in which the culture pH gradually increased from standard 6.2 to 7.0 (ΔPh = 0.2-0.3), and cells were maintained at each pH value for 2-3 weeks until a constant growth rate and a cell viability over 95% were observed. These adapted cells enabled an increase in cell-specific HA productivity up to three-fold and volumetric HA titer of up to four-fold as compared to non-adapted cells. Of note, the adaptation process is the element driving increased specific HA productivity as a pH shift alone was inefficient at improving productivities. The production of HA-VLPs in adapted cells was successfully demonstrated at the bioreactor scale. The produced HA-VLPs show the typical size and morphology of influenza VLPs, thus confirming the null impact of the adaptation process and neutral culture pH on the quality of HA-VLPs produced. This work strengthens the potential of ALE as a bioprocess engineering strategy to improve the production of influenza HA-VLPs in insect High Five cells.
已证明使用非标准培养条件可有效提高不同细胞宿主中的细胞性能和重组蛋白产量。然而，通过适应性实验室进化（ALE）建立高产细胞群体的研究较少，特别是对于昆虫细胞。在本研究中，通过ALE成功使昆虫High Five细胞适应在中性培养pH（7.0）下生长，以提高展示流感血凝素（HA）的病毒样颗粒（VLP）的产量。采用逐步方法进行适应过程，其中培养pH从标准的6.2逐渐增加到7.0（ΔPh = 0.2 - 0.3），细胞在每个pH值下维持2 - 3周，直到观察到恒定的生长速率和超过95%的细胞活力。与未适应的细胞相比，这些适应的细胞使细胞特异性HA生产力提高了三倍，体积HA滴度提高了四倍。值得注意的是，适应过程是推动细胞特异性HA生产力提高的因素，因为仅pH变化在提高生产力方面效率低下。在生物反应器规模上成功证明了在适应细胞中生产HA-VLP。所生产的HA-VLP显示出流感VLP的典型大小和形态，从而证实了适应过程和中性培养pH对所生产的HA-VLP质量没有影响。这项工作加强了ALE作为一种生物过程工程策略在提高昆虫High Five细胞中流感HA-VLP产量方面的潜力。
