Konakovsky Viktor, Clemens Christoph, Müller Markus Michael, Bechmann Jan, Berger Martina, Schlatter Stefan, Herwig Christoph
Institute of Chemical Engineering, Division of Biochemical Engineering, Vienna University of Technology, Gumpendorfer Strasse 1A 166-4, 1060 Vienna, Austria.
Boehringer Ingelheim Pharma GmbH & Co. KG Dep. Bioprocess Development, Biberach, Germany.
Bioengineering (Basel). 2016 Jan 11;3(1):5. doi: 10.3390/bioengineering3010005.
Biomass and cell-specific metabolic rates usually change dynamically over time, making the "feed according to need" strategy difficult to realize in a commercial fed-batch process. We here demonstrate a novel feeding strategy which is designed to hold a particular metabolic state in a fed-batch process by adaptive feeding in real time. The feed rate is calculated with a transferable biomass model based on capacitance, which changes the nutrient flow stoichiometrically in real time. A limited glucose environment was used to confine the cell in a particular metabolic state. In order to cope with uncertainty, two strategies were tested to change the adaptive feed rate and prevent starvation while in limitation: (i) inline pH and online glucose concentration measurement or (ii) inline pH alone, which was shown to be sufficient for the problem statement. In this contribution, we achieved within a defined target range. The direct benefit was two-fold: the lactic acid profile was improved and pH could be kept stable. Multivariate Data Analysis (MVDA) has shown that pH influenced lactic acid production or consumption in historical data sets. We demonstrate that a low pH (around 6.8) is not required for our strategy, as glucose availability is already limiting the flux. On the contrary, we boosted glycolytic flux in glucose limitation by setting the pH to 7.4. This new approach led to a yield of lactic acid/glucose (Y L/G) around zero for the whole process time and high titers in our labs. We hypothesize that a higher carbon flux, resulting from a higher pH, may lead to more cells which produce more product. The relevance of this work aims at feeding mammalian cell cultures safely in limitation with a desired metabolic flux range. This resulted in extremely stable, low glucose levels, very robust pH profiles without acid/base interventions and a metabolic state in which lactic acid was consumed instead of being produced from day 1. With this contribution, we wish to extend the basic repertoire of available process control strategies, which will open up new avenues in automation technology and radically improve process robustness in both process development and manufacturing.
生物量和细胞特异性代谢率通常会随时间动态变化，这使得“按需进料”策略在商业化补料分批培养过程中难以实现。我们在此展示了一种新颖的进料策略，该策略旨在通过实时自适应进料在补料分批培养过程中维持特定的代谢状态。进料速率通过基于电容的可转移生物量模型进行计算，该模型实时按化学计量比改变营养物质流。使用有限葡萄糖环境将细胞限制在特定代谢状态。为应对不确定性，测试了两种策略来改变自适应进料速率并在受限状态下防止饥饿：（i）在线pH和在线葡萄糖浓度测量，或（ii）仅在线pH测量，结果表明后者足以解决该问题。在本研究中，我们在定义的目标范围内实现了……。直接益处有两方面：乳酸谱得到改善，pH可保持稳定。多变量数据分析（MVDA）表明，在历史数据集中pH会影响乳酸的产生或消耗。我们证明，对于我们的策略而言，并不需要低pH（约6.8），因为葡萄糖可用性已经限制了通量。相反，我们通过将pH设置为7.4来提高葡萄糖受限情况下的糖酵解通量。这种新方法在整个过程时间内使乳酸/葡萄糖产率（Y L/G）约为零，并在我们实验室中实现了高滴度。我们推测，较高的pH导致更高的碳通量，可能会产生更多产生更多产物的细胞。这项工作的意义在于在限制条件下以所需的代谢通量范围安全地补料培养哺乳动物细胞。这导致了极其稳定的低葡萄糖水平、无需酸碱干预的非常稳健的pH谱以及从第一天起乳酸被消耗而非产生的代谢状态。通过本研究，我们希望扩展可用过程控制策略的基本方法，这将为自动化技术开辟新途径，并从根本上提高过程开发和制造中的过程稳健性。
