Konstantinov Konstantin, Goudar Chetan, Ng Maria, Meneses Renato, Thrift John, Chuppa Sandy, Matanguihan Cary, Michaels Jim, Naveh David
Bayer HealthCare, Biological Products Division, 800 Dwight Way, P.O.Box 1986, Berkeley, CA 94710, USA.
Adv Biochem Eng Biotechnol. 2006;101:75-98. doi: 10.1007/10_016.
High product titer is considered a strategic advantage of fed-batch over perfusion cultivation mode. The titer difference has been experimentally demonstrated and reported in the literature. However, the related theoretical aspects and strategies for optimization of perfusion processes with respect to their fed-batch counterparts have not been thoroughly explored. The present paper introduces a unified framework for comparison of fed-batch and perfusion cultures, and proposes directions for improvement of the latter. The comparison is based on the concept of "equivalent specific perfusion rate", a variable that conveniently bridges various cultivation modes. The analysis shows that development of economically competitive perfusion processes for production of stable proteins depends on our ability to dramatically reduce the dilution rate while keeping high cell density, i.e., operating at low specific perfusion rates. Under these conditions, titer increases significantly, approaching the range of fed-batch titers. However, as dilution rate is decreased, a limit is reached below which performance declines due to poor growth and viability, specific productivity, or product instability. To overcome these limitations, a strategy referred to as "push-to-low" optimization has been developed. This approach involves an iterative stepwise decrease of the specific perfusion rate, and is most suitable for production of stable proteins where increased residence time does not compromise apparent specific productivity or product quality. The push-to-low approach was successfully applied to the production of monoclonal antibody against tumor necrosis factor (TNF). The experimental results followed closely the theoretical prediction, providing a multifold increase in titer. Despite the medium improvement, reduction of the specific growth rate along with increased apoptosis was observed at low specific perfusion rates. This phenomenon could not be explained with limitation or inhibition by the known nutrients and metabolites. Even further improvement would be possible if the cause of apoptosis were understood. In general, a strategic target in the optimization of perfusion processes should be the decrease of the cell-specific perfusion rate to below 0.05 nL/cell/day, resulting in high, batch-like titers. The potential for high titer, combined with high volumetric productivity, stable performance over many months, and superior product/harvest quality, make perfusion processes an attractive alternative to fed-batch production, even in the case of stable proteins.
高产品滴度被认为是补料分批培养相对于灌注培养模式的一个战略优势。滴度差异已通过实验得到证明，并在文献中有报道。然而，关于灌注过程相对于其补料分批对应过程的相关理论方面以及优化策略尚未得到充分探索。本文介绍了一个用于比较补料分批培养和灌注培养的统一框架，并提出了改进后者的方向。该比较基于“等效比灌注速率”的概念，这是一个方便地连接各种培养模式的变量。分析表明，开发具有经济竞争力的用于生产稳定蛋白质的灌注过程取决于我们在保持高细胞密度的同时大幅降低稀释率的能力，即在低比灌注速率下操作。在这些条件下，滴度显著增加，接近补料分批培养的滴度范围。然而，随着稀释率的降低，会达到一个极限，低于该极限时，由于生长不良、活力下降、比生产率或产品不稳定性，性能会下降。为了克服这些限制，已开发出一种称为“推至低”优化的策略。这种方法涉及比灌注速率的迭代逐步降低，并且最适合于生产稳定蛋白质，其中增加的停留时间不会损害表观比生产率或产品质量。“推至低”方法已成功应用于抗肿瘤坏死因子（TNF）单克隆抗体的生产。实验结果与理论预测密切相符，使滴度提高了数倍。尽管培养基有所改进，但在低比灌注速率下仍观察到比生长速率降低以及凋亡增加。这种现象无法用已知营养物质和代谢产物的限制或抑制来解释。如果能理解凋亡的原因，甚至可能实现进一步的改进。一般来说，灌注过程优化的一个战略目标应该是将细胞比灌注速率降低到0.05 nL/细胞/天以下，并产生高的、类似批次的滴度。高滴度的潜力，加上高体积生产率、数月的稳定性能以及优异的产品/收获质量，使得灌注过程即使在生产稳定蛋白质的情况下，也是补料分批生产的一个有吸引力的替代方案。
