Maranga Luis, Cunha António, Clemente João, Cruz Pedro, Carrondo Manuel J T
Instituto de Biologia Experimental e Tecnológica/Instituto de Tecnologia Química e Biológica IBET/ITQB, Apartado 12, Oeiras P-2781-901, Portugal.
J Biotechnol. 2004 Jan 8;107(1):55-64. doi: 10.1016/j.jbiotec.2003.09.012.
The baculovirus-insect cells expression system was used for the production of self-forming Porcine parvovirus (PPV) like particles (virus-like particles, VLPs) in serum-free medium. At 2l bioreactor scale an efficient production was achieved by infecting the culture at a concentration of 1.5 x 10(6)cells/ml using a low multiplicity of infection of 0.05 pfu per cell. In a continuous bioreactor, it was shown that the uninfected insect cells were not sensitive to local shear stress values up to 2.25 N/m2 at high Reynolds numbers (1.5 x 10(4)) in sparging conditions. Uninfected insect cells can be grown at scaled-up bioreactor at high agitation and sparging rates as long as vortex formation is avoided and bubble entrapment is minimized. An efficient process scale-up to 25 l bioreactor was made using constant shear stress criteria for scale-up. The kinetics of baculovirus infection at low multiplicity of infection, either at different cell concentration or at different scales, are very reproducible, despite the different turbulence conditions present in the bioreactor milieu. The results suggest that the infection kinetics is controlled by the rate of baculovirus-cell receptor attachment and is independent of the bioreactor hydrodynamic conditions. Furthermore, the achieved specific and volumetric productivities were higher at the 25 l scale when compared to the smaller scale bioreactor. Different rates of cell lysis after infection were observed and seem to fully explain both the shift in optimal harvest time and the increase in cell specific productivity. The results emphasize the importance of integrated strategies and engineering concepts in process development at bioreactor stage with the baculovirus insect cell system.
杆状病毒-昆虫细胞表达系统用于在无血清培养基中生产自组装的猪细小病毒（PPV）样颗粒（病毒样颗粒，VLPs）。在2L生物反应器规模下，通过以1.5×10⁶个细胞/ml的浓度感染培养物，并使用每个细胞0.05 pfu的低感染复数，实现了高效生产。在连续生物反应器中，结果表明，在鼓泡条件下，当雷诺数较高（1.5×10⁴）时，未感染的昆虫细胞对高达2.25 N/m²的局部剪切应力值不敏感。只要避免形成漩涡并尽量减少气泡截留，未感染的昆虫细胞就可以在放大的生物反应器中以高搅拌和鼓泡速率生长。使用恒定剪切应力标准进行放大，实现了高效放大至25L生物反应器。尽管生物反应器环境中存在不同的湍流条件，但在低感染复数下，无论细胞浓度不同还是规模不同，杆状病毒感染的动力学都具有很高的可重复性。结果表明，感染动力学由杆状病毒-细胞受体附着速率控制，与生物反应器的流体动力学条件无关。此外，与较小规模的生物反应器相比，在25L规模下获得的比生产率和体积生产率更高。观察到感染后不同的细胞裂解速率，这似乎充分解释了最佳收获时间的变化和细胞比生产率的提高。结果强调了在杆状病毒昆虫细胞系统的生物反应器阶段，综合策略和工程概念在工艺开发中的重要性。
