High-density mammalian cell cultures in stirred-tank bioreactor without external pH control
Introduction
Importance of pH on mammalian cell growth, metabolism, and product formation has been extensively studied in different operational modes (Borys et al., 1993, Miller et al., 1988, Ozturk and Palsson, 1991, Yoon et al., 2005). In a typical bioreactor system, a feedback control loop is required to maintain pH at the setpoint that varies among cell lines and processes. When pH is lower than the target value, a base pump will be triggered to deliver base (e.g., NaOH, Na2CO3, or NaHCO3) to bring pH up. When pH is higher than the target value, an acid pump or CO2 valve will be turned on to deliver either acid (e.g., HCl or lactic acid) or CO2 to lower pH. It is common to use CO2 for pH control in combination with a bicarbonate-buffering system in mammalian cell cultures. When the base pump or CO2 valve will be triggered is determined by the tuning parameters defined in the control loop. To avoid the negative impact from pH gradients created by base addition, optimization of pH control loop, tuning parameters, and location of base addition is critical (Langheinrich and Nienow, 1999, Nienow et al., 1996, Osman et al., 2002). However, this sometimes is a challenge in large-scale bioreactors since facility shutdown is often required for such activities. Minimum to no base addition is often viewed as an indicator for a good process, while a process that requires large amount of base addition could be problematic since base addition is typically related to high pCO2 and/or high lactate accumulations (Charaniya et al., 2010, Nienow, 2006).
Controlling pH can be more challenging in high-density cell cultures and process scale-up. For fed-batch cultures at high densities (e.g., 20–30 × 106 cells/mL), high pCO2 build-up in large-scale bioreactors could compromise process performance due to the additional base required and the accompanied osmolality increase (Nienow, 2006). Mixing difference between bioreactor configurations or scales could also cause issues during base addition, which could further impact product titer (Sieblist et al., 2015). For perfusion cultures, medium is continuously exchanged out and new balance has to be established continuously for pH control, which calls for more base for pH control. A few studies have evaluated the effect of different bases (NaOH, Na2CO3, and NaHCO3) on CO2 accumulation and cell viability in perfusion cultures (Goudar et al., 2007, Matanguihan et al., 2001, Ozturk, 1996). Maintaining constant glucose and lactate levels in perfusion cultures through online glucose/lactate monitoring has been shown to be effective in reducing or eliminating base addition (Konstantinov et al., 1996, Ozturk et al., 1997). To achieve such goals, additional real-time glucose/lactate monitoring equipment will have to be used, and this could add complexity in process development. Recently, a few studies have shown the possibility of maintaining good cell growth without pH and DO controls through optimizing engineering parameters of either Wave bioreactors or orbitally shaken bioreactors (OSR) for 7–14 days (Tissot et al., 2011, Yuk et al., 2011). However, none of them demonstrated desired pH control for long-term protein production.
In this study, we explored the possibility of cultivating Chinese hamster ovary (CHO) cells with minimum or no pH control, in both high-density fed-batch (14 days at both 3 L and 200 L scales) and perfusion cultures (up to 40 days at 3 L scale). Typically, pH is determined by the lactate and pCO2 levels when no base is added in a pre-defined mammalian culture process. Thus, lactate and pCO2 could be potentially leveraged to maintain a desired culture pH without the need of external pH control loops. In such case, studies on the impact of different bases on cell viability and CO2 accumulation at different scales could be avoided since base addition will not be required. For fed-batch cultures, process performances at different controlling pH ranges were evaluated. High-density cultures (21.7 ± 1.8 × 106 cells/mL) in the 3 L and 200 L bioreactors were evaluated to demonstrate the pCO2 difference from different bioreactor scales and sparger configurations, and the potential impact of that on culture pH. For perfusion cultures, the effectiveness of maintaining bioreactor pH in two distinguishably different processes with steady state cell densities at 42.5 ± 3.3 or 68.3 ± 6.0 × 106 cells/mL without CO2 sparging and base addition was assessed. The impact of antifoam addition on CO2 stripping and the impact of pH fluctuations on product quality attributes were also discussed.
Section snippets
Cell line and inoculum expansion
A recombinant CHO cell line developed in-house was used in the study. It was a glutamine synthetase (GS) cell line designed to produce a monoclonal antibody. The inoculum train started from vial thaw and expanded in shake flasks and Wave bioreactors (GE Healthcare, Uppsala, Sweden). Both shake flasks and Wave bioreactors were maintained at 5% CO2 and 36.5 °C.
Batch cultures (15 mL)
Microbioreactors (ambr15™, Sartorius Stedim, Germany) were used in the initial pH evaluation. The microbioreactors were inoculated at a
Impact of pCO2 and lactate on pH
It is well-known that pCO2, lactate, and base consumption are interlinked factors determining the pH of a bicarbonate-buffered system used in mammalian cell cultures (Abu-Absi et al., 2014, Gramer and Ogorzalek, 2007). During the course of cell growth, pH will undergo changes due to the production of CO2 and lactate. This is shown in the following equations:
Generation of both CO2 and lactate would make medium acidic.
Conclusions
In this study, we demonstrated the feasibility of high-density cell culture processes with minimum or no pH control, in both fed-batch mode for 14 days and perfusion mode for up to 40 days. Culture pH was determined by the lactate and pCO2 levels, instead of the external control using CO2 sparging and base addition. In fed-batch cultures, comparable results were obtained at different upper pH limits (7.05, 7.30, 7.45, or 7.65) with a lower pH limit of 6.75, suggesting that no pH or only minimum
Acknowledgements
We would like to thank Michael Caruso, Alejandro Baloco, Debra Lutz, James Jimenze, Jack Cardoso, and D’Juan Gibson for media preparation and bioreactor operations; Linda Hoshan for assistance in ambr15™ operations; Rubin Jiang for generating data in Fig. S3; Elizabeth Wu, Heera Khan, Michael Rauscher, Sonja Battle, and Yan Peng for purification support and quality analysis. We would also like to acknowledge the constructive review of the manuscript by T. Craig Seamans and David Roush.
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