Cruz P E, Cunha A, Peixoto C C, Clemente J, Moreira J L, Carrondo M J
Instituto de Biologia Experimental e Tecnológica/Instituto de Tecnologia Química e Biológica IBET/ITQB, Apartado 12, P-2780 Oeiras, Portugal.
Biotechnol Bioeng. 1998 Nov 20;60(4):408-18.
In this work the maximal operational hydrodynamic conditions (agitation and aeration rate) that cause no adverse effect in Sf-9 cells growth in SF900II serum-free medium were determined. Shear stresses higher than 1 N m-2 and aeration rates higher than 0.04 vvm affect cell growth and when these conditions increase to 1.5 N m-2 and 0.11 vvm, cell growth is completely inhibited with significant cell morphology changes and a strong decrease in viability. Although the pO2 did not show a significant effect upon cell growth in the range from 10 to 50%, cell infection and specific productivity were dramatically affected. The production was optimal at a pO2 of 25% with decreases higher than 50% being observed when the pO2 decreased to 10 or increased to 50%. The maximum product quality, i.e., the percentage of product in the form of high molecular weight particles, is not coincident with maximum product titer. Although the highest Pr55gag particle titer was obtained at 96 hours post infection (hpi) and at pO2 of 25%, the best product quality (defined by gel filtration chromatography and Western immunoblot) was obtained at 48 hpi, independently of the pO2 used. The effect of overcritical conditions upon productivity was also studied. As obtained for cell growth, cell infection is affected by shear stresses above 1 N m-2 and by aeration rates higher than 0.04 vvm, with decreases in Pr55gag particle titer higher than 70%, even when the overcritical values are still far from the limit at which cell death occurs. The results obtained and the optimization strategy used allowed the maximization of the oxygen supply without damaging the cells, with important consequences on the scale-up of a production process involving this insect cell/baculovirus expression system.
在本研究中，确定了在SF900II无血清培养基中对Sf-9细胞生长无不利影响的最大操作流体动力学条件（搅拌和通气速率）。高于1 N m-2的剪切应力和高于0.04 vvm的通气速率会影响细胞生长，当这些条件增加到1.5 N m-2和0.11 vvm时，细胞生长完全受到抑制，细胞形态发生显著变化，活力大幅下降。尽管在10%至50%的范围内，pO2对细胞生长未显示出显著影响，但细胞感染和比生产率受到了显著影响。在pO2为25%时产量最佳，当pO2降至10%或升至50%时，产量下降超过50%。最大产品质量，即以高分子量颗粒形式存在的产品百分比，与最大产品滴度不一致。尽管在感染后96小时（hpi）和pO2为25%时获得了最高的Pr55gag颗粒滴度，但在48 hpi时获得了最佳产品质量（通过凝胶过滤色谱和Western免疫印迹定义），与所用的pO2无关。还研究了超临界条件对生产率的影响。与细胞生长情况一样，高于1 N m-2的剪切应力和高于0.04 vvm的通气速率会影响细胞感染，Pr55gag颗粒滴度下降超过70%，即使超临界值仍远未达到细胞死亡的极限。所获得的结果和采用的优化策略使得在不损害细胞的情况下最大限度地供应氧气，这对涉及该昆虫细胞/杆状病毒表达系统的生产过程放大具有重要意义。
