Fernandes Bárbara, Correia Ricardo, Alves Paula M, Roldão António
IBET-Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal.
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal.
Front Bioeng Biotechnol. 2022 Jun 29;10:917746. doi: 10.3389/fbioe.2022.917746. eCollection 2022.
Protein production processes based on stable insect cell lines require intensification to be competitive with the insect cell-baculovirus expression vector system (IC-BEVS). High cell density (HCD) cultures operate continuously, capable of maintaining specific production rates for extended periods of time which may lead to significant improvements in production yields. However, setting up such processes is challenging (e.g., selection of cell retention device and optimization of dilution rate), often demanding the manipulation of large volumes of culture medium with associated high cost. In this study, we developed a process for continuous production of Gag virus-like particles (VLP) pseudotyped with a model membrane protein (influenza hemagglutinin, HA) at HCD using stable insect cells adapted to low culture temperature. The impact of the cell retention device (ATF vs. TFF) and cell-specific perfusion rate (CSPR) on cell growth and protein expression kinetics was evaluated. Continuous production of Gag-HA VLPs was possible using both retention devices and CSPR of 0.04 nL/cell.d; TFF induces higher cell lysis when compared to ATF at later stages of the process (k = 0.009 vs. 0.005 h, for TFF and ATF, respectively). Reducing CSPR to 0.01-0.02 nL/cell.d using ATF had a negligible impact on specific production rates (r = 72-68 titer/10 cell.h and r = 12-11 pg/10 cell.h in all CSPR) and on particle morphology (round-shaped structures displaying HA spikes on their surface) and size distribution profile (peaks at approximately 100 nm). Notably, at these CSPRs, the amount of p24 or HA formed per volume of culture medium consumed per unit of process time increases by up to 3-fold when compared to batch and perfusion operation modes. Overall, this work demonstrates the potential of manipulating CSPRs to intensify the continuous production of Gag-HA VLPs at HCD using stable insect cells to make them an attractive alternative platform to IC-BEVS.
基于稳定昆虫细胞系的蛋白质生产工艺需要强化，以使其能与昆虫细胞-杆状病毒表达载体系统（IC-BEVS）竞争。高细胞密度（HCD）培养可连续运行，能够长时间维持特定生产率，这可能会显著提高产量。然而，建立这样的工艺具有挑战性（例如，细胞截留装置的选择和稀释率的优化），通常需要处理大量培养基，成本高昂。在本研究中，我们开发了一种在HCD条件下，使用适应低温培养的稳定昆虫细胞连续生产以模型膜蛋白（流感血凝素，HA）假型化的Gag病毒样颗粒（VLP）的工艺。评估了细胞截留装置（交替切向流过滤（ATF）与切向流过滤（TFF））和细胞特异性灌注速率（CSPR）对细胞生长和蛋白质表达动力学的影响。使用两种截留装置和0.04 nL/细胞·天的CSPR都可以连续生产Gag-HA VLP；在工艺后期，与ATF相比，TFF诱导更高的细胞裂解（TFF和ATF的k分别为0.009 h⁻¹和0.005 h⁻¹）。使用ATF将CSPR降低到0.01 - 0.02 nL/细胞·天对特定生产率（所有CSPR下r = 72 - 68效价/10⁶细胞·小时和r = 12 - 11 pg/10⁶细胞·小时）以及颗粒形态（表面显示HA刺突的圆形结构）和尺寸分布曲线（峰值约为100 nm）的影响可忽略不计。值得注意的是，在这些CSPR下，与分批和灌注操作模式相比，单位工艺时间内每消耗单位体积培养基形成的p24或HA量增加了高达3倍。总体而言，这项工作证明了通过控制CSPR来强化使用稳定昆虫细胞在HCD条件下连续生产Gag-HA VLP的潜力，使其成为IC-BEVS有吸引力的替代平台。
