Development, characterization, and application of a 2‐Compartment system to investigate the impact of pH inhomogeneities in large‐scale CHO‐based processes

Paul, Katrin; Böttinger, Katharina; Mitic, Bernd M.; Scherfler, Georg; Posch, C.; Behrens, Dirk; Huber, Christian G.; Herwig, Christoph

doi:10.1002/elsc.202000009

Cited by 7 publications

(10 citation statements)

References 45 publications

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“…It has previously been shown that the exposure of cells to amplitudes of only 0.4 pH units already negatively impacts process performance. Furthermore, the number of exposed cells or the amount of added base was identified as the main culprits for impaired process performance [23]. Here the impact of smaller pH amplitudes (magnitude of 0.05 units) and of a narrow pH control without the introduction of amplitudes are investigated for cell lines A and B.…”

Section: Resultsmentioning

confidence: 99%

“…Glucose was fed continually after its concentration dropped below 2 g L −1 to reestablish glucose levels of 2 g L −1 . The 2‐CSs were setup according to previously published work [23]. They consist of a bioreactor vessel with added outlet at the bottom and a bypass for the recirculation of the cells.…”

Section: Methodsmentioning

confidence: 99%

“…However, base addition at large‐scale would cause frequent pH excursions for only short periods of time [4,21,22]. Recent work has shown that the exposure of CHO cells to reoccurring pH amplitudes of an average magnitude of 7.3 already impairs process performance and inhibits the lactate metabolic shift [23]. Due to inherent differences of the various CHO cell lineages, the response to pH inhomogeneity may however vary from cell line to cell line [24].…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, this study focuses on the investigation of differences between the cell line lineage specific response to pH inhomogeneity. Since previous work has shown a negative impact on process performance, when cells were introduced to pH amplitudes of 0.4 pH units [23], this study investigates amplitudes of a maximal magnitude of 0.1 pH units. Both cell lines were exposed to similar pH excursions in 2‐CSs, where base addition started at the same time point for all experiments.…”

Section: Introductionmentioning

confidence: 99%

“…Both cell lines were exposed to similar pH excursions in 2‐CSs, where base addition started at the same time point for all experiments. However, base addition started later in the previous study [23], therefore the impact of the starting time point of pH correction on process performance was also investigated. Furthermore, submerse base addition has been suggested as a solution to gradients occurring at large‐scale by improving mixing [4,25].…”

Section: Introductionmentioning

confidence: 99%

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Investigation of cell line specific responses to pH inhomogeneity and consequences for process design

Paul

Hartmann

Posch³

et al. 2020

Engineering in Life Sciences

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With increasing bioreactor volumes, the mixing time of the reactor increases as well, which creates an inhomogeneous environment for the cells. This can result in impaired process performance in large‐scale production reactors. Particularly the addition of base through the reactor headspace can be problematic, since it creates an area, where cells are repeatedly exposed to an increased pH. The aim of this study is to simulate this large‐scale phenomenon at lab‐scale and investigate its impact. Two different cell lines were exposed to pH amplitudes of a maximal magnitude of 0.05 units (pH of 6.95). Both cell lines showed similar responses, like decreased viable cell counts, but unaffected lactate levels. However, cell line B showed an initially increased specific productivity in response to the introduced amplitudes, whereas cell line A showed a consistently lower specific productivity. Furthermore, the time point at which base addition is started influences the impact, which pH amplitudes have on process performance. When pH control was started earlier in the process, maximal viable cell counts decreased and the lactate metabolic shift was less pronounced. These results show that the potential negative impact of pH amplitudes can be minimized by strategic process design.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Investigation of cell line specific responses to pH inhomogeneity and consequences for process design

Paul

Hartmann

Posch³

et al. 2020

Engineering in Life Sciences

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Mimicking CHO large‐scale effects in the single multicompartment bioreactor: A new approach to access scale‐up behavior

Gaugler,

Hofmann,

Schlüter

et al. 2024

Biotech & Bioengineering

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During the scale‐up of biopharmaceutical production processes, insufficiently predictable performance losses may occur alongside gradients and heterogeneities. To overcome such performance losses, tools are required to explain, predict, and ultimately prohibit inconsistencies between laboratory and commercial scale. In this work, we performed CHO fed‐batch cultivations in the single multicompartment bioreactor (SMCB), a new scale‐down reactor system that offers new access to study large‐scale heterogeneities in mammalian cell cultures. At volumetric power inputs of 20.4–1.5 W m−3, large‐scale characteristics like long mixing times and dissolved oxygen (DO) heterogeneities were mimicked in the SMCB. Compared to a reference bioreactor (REFB) set‐up, the conditions in the SMCB provoked an increase in lactate accumulation of up to 87%, an increased glucose uptake, and reduced viable cell concentrations in the stationary phase. All are characteristic for large‐scale performance. The unique possibility to distinguish between the effects of changing power inputs and observed heterogeneities provided new insights into the potential reasons for altered product quality attributes. Apparently, the degree of galactosylation in the evaluated glycan patterns changed primarily due to the different power inputs rather than the provoked heterogeneities. The SMCB system could serve as a potent tool to provide new insights into scale‐up behavior and to predict cell line‐specific drawbacks at an early stage of process development.

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An industrial perspective on metabolic responses of Penicillium chrysogenum to periodic dissolved oxygen feast‐famine cycles in a scale‐down system

Wang,

Yang,

Haringa

et al. 2024

Biotech & Bioengineering

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While traveling through different zones in large‐scale bioreactors, microbes are most likely subjected to fluctuating dissolved oxygen (DO) conditions at the timescales of global circulation time. In this study, to mimic industrial‐scale spatial DO gradients, we present a scale‐down setup based on dynamic feast/famine regime (150 s) that leads to repetitive cycles with rapid changes in DO availability in glucose‐limited chemostat cultures of Penicillium chrysogenum. Such DO feast/famine regime induced a stable and repetitive pattern with a reproducible metabolic response in time, and the dynamic response of intracellular metabolites featured specific differences in terms of both coverage and magnitude in comparison to other dynamic conditions, for example, substrate feast/famine cycles. Remarkably, intracellular sugar polyols were considerably increased as the hallmark metabolites along with a dynamic and higher redox state nicotinamide adenine dinucleotide hydrogen/nicotinamide adenine dinucleotide of the cytosol. Despite the increased availability of NADPH for penicillin production under the oscillatory DO conditions, this positive effect may be counteracted by the decreased adenosine triphosphate supply. Moreover, it is interesting to note that not only the penicillin productivity was reduced under such oscillating DO conditions, but also that of the unrecyclable byproduct ortho‐hydroxyphenyl acetic acid and degeneration of penicillin productivity. Furthermore, dynamic flux profiles showed the most pronounced variations in central carbon metabolism, amino acid (AA) metabolism, energy metabolism and fatty acid metabolism upon the DO oscillation. Taken together, the metabolic responses of P. chrysogenum to DO gradients reported here are important for elucidating metabolic regulation mechanisms, improving bioreactor design and scale‐up procedures as well as for constructing robust cell strains to cope with heterogenous industrial culture conditions.

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Development, characterization, and application of a 2‐Compartment system to investigate the impact of pH inhomogeneities in large‐scale CHO‐based processes

Cited by 7 publications

References 45 publications

Investigation of cell line specific responses to pH inhomogeneity and consequences for process design

Investigation of cell line specific responses to pH inhomogeneity and consequences for process design

Mimicking CHO large‐scale effects in the single multicompartment bioreactor: A new approach to access scale‐up behavior

An industrial perspective on metabolic responses of Penicillium chrysogenum to periodic dissolved oxygen feast‐famine cycles in a scale‐down system

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