Department of Chemical Engineering, University of Colorado, Boulder, CO, 80309-0424, U.S.A.
Cytotechnology. 1998 Nov;28(1-3):73-80. doi: 10.1023/A:1008069312131.
Protein synthesis in mammalian cells can be observed in two strikingly different patterns: 1) production of monoclonal antibodies in hybridoma cultures is typically inverse growth associated and 2) production of most therapeutic glycoproteins in recombinant mammalian cell cultures is found to be growth associated. Production of monoclonal antibodies has been easily maximized by culturing hybridoma cells at very low growth rates in high cell density fed- batch or perfusion bioreactors. Applying the same bioreactor techniques to recombinant mammalian cell cultures results in drastically reduced production rates due to their growth associated production kinetics. Optimization of such growth associated production requires high cell growth conditions, such as in repeated batch cultures or chemostat cultures with attendant excess biomass synthesis. Our recent research has demonstrated that this growth associated production in recombinant Chinese hamster ovary (CHO) cells is related to the S (DNA synthesis)-phase specific production due to the SV40 early promoter commonly used for driving the foreign gene expression. Using the stably transfected CHO cell lines synthesizing an intracellular reporter protein under the control of SV40 early promoter, we have recently demonstrated in batch and continuous cultures that the product synthesis is growth associated. We have now replaced this S-phase specific promoter in new expression vectors with the adenovirus major late promoter which was found to be active primarily in the G1-phase and is expected to yield the desirable inverse growth associated production behavior. Our results in repeated batch cultures show that the protein synthesis kinetics in this resulting CHO cell line is indeed inverse growth associated. Results from continuous and high cell density perfusion culture experiments also indicate a strong inverse growth associated protein synthesis. The bioreactor optimization with this desirable inverse growth associated production behavior would be much simpler than bioreactor operation for cells with growth associated production.
1）杂交瘤培养物中单克隆抗体的产生通常与逆生长相关，2）重组哺乳动物细胞培养物中大多数治疗性糖蛋白的产生与生长相关。通过在高细胞密度分批或灌注生物反应器中以非常低的生长速率培养杂交瘤细胞，很容易使单克隆抗体的产生最大化。将相同的生物反应器技术应用于重组哺乳动物细胞培养物会导致由于其与生长相关的生产动力学而导致生产速率急剧降低。由于与生长相关的生产需要高细胞生长条件，例如在重复分批培养物或恒化器培养物中，伴随有过多的生物质合成，因此需要对这种与生长相关的生产进行优化。我们最近的研究表明，这种与生长相关的生产与 SV40 早期启动子通常用于驱动外源基因表达有关，与重组中国仓鼠卵巢（CHO）细胞中的 S（DNA 合成）期特异性生产有关。使用受 SV40 早期启动子控制的稳定转染 CHO 细胞系合成细胞内报告蛋白，我们最近在分批和连续培养中证明，产物合成与生长相关。我们现在已经用腺病毒主要晚期启动子替换了这些在新表达载体中的 S 期特异性启动子，该启动子主要在 G1 期活跃，预计会产生所需的与生长相反的相关生产行为。我们在重复分批培养物中的结果表明，该 CHO 细胞系中的蛋白质合成动力学确实与生长相反。连续和高细胞密度灌注培养实验的结果也表明强烈的与生长相反的蛋白质合成。与具有生长相关的生产的细胞相比，这种理想的与生长相反的生产行为的生物反应器优化要简单得多。
