A new strategy for fed-batch process control of HEK293 cell cultures based on alkali buffer addition monitoring: comparison with O.U.R. dynamic method

Martínez-Monge, I.; Comas, P.; Triquell, J.; Lecina, Martí; Casablancas, Antoni; Cairó, Jordi J.

doi:10.1007/s00253-018-9388-4

Cited by 17 publications

(14 citation statements)

References 37 publications

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“…This method is based on interrupting the air inflow to the bioreactor for a few minutes; then, the dissolved oxygen concentration decreases due to the respiratory activity of the cells and to the oxygen desorption from the liquid phase to the bioreactor headspace. The specific methodology used in this work is detailed in the literature [31,32].…”

Section: Oxygen Uptake Rate (Our)mentioning

confidence: 99%

The Effect of the Expression of the Antiapoptotic BHRF1 Gene on the Metabolic Behavior of a Hybridoma Cell Line

et al. 2021

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One of the most important limitations of mammalian cells-based bioprocesses, and particularly hybridoma cell lines, is the accelerated metabolism related to glucose and glutamine consumption. The high uptake rates of glucose and glutamine (i.e., the main sources of carbon, nitrogen and energy) lead to the production and accumulation of large amounts of lactate and ammonia in culture broth. Lactate and/or ammonia accumulation, together with the depletion of the main nutrients, are the major causes of apoptosis in hybridoma cell cultures. The KB26.5 hybridoma cell line, producing an IgG3, was engineered with BHRF1 (KB26.5-BHRF1), an Epstein–Barr virus-encoded early protein homologous to the antiapoptotic protein Bcl-2, with the aim of protecting the hybridoma cell line from apoptosis. Surprisingly, besides achieving effective protection from apoptosis, the expression of BHRF1 modified the metabolism of the hybridoma cell line. Cell physiology and metabolism analyses of the original KB26.5 and KB26.5-BHRF1 revealed an increase of cell growth rate, a reduction of glucose and glutamine consumption, as well as a decrease in lactate secretion in KB26.5-BHRF1 cells. A flux balance analysis allowed us to quantify the intracellular fluxes of both cell lines. The main metabolic differences were identified in glucose consumption and, consequently, the production of lactate. The lactate production flux was reduced by 60%, since the need for NADH regeneration in the cytoplasm decreased due to a more than 50% reduction in glucose uptake. In general terms, the BHRF1 engineered cell line showed a more efficient metabolism, with an increase in biomass volumetric productivity under identical culture conditions.

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Section: Oxygen Uptake Rate (Our)mentioning

confidence: 99%

The Effect of the Expression of the Antiapoptotic BHRF1 Gene on the Metabolic Behavior of a Hybridoma Cell Line

et al. 2021

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“…Different examples can be found in the application of softsensors to estimate variables like viable cell density (VCD) and metabolites concentration, as permittivity and turbidity (Kroll et al, 2017), or oxygen uptake rate (OUR) (Martínez-Monge et al, 2019b). OUR is one of the most feasible variables to accurately estimate the cells' demand.…”

Section: Introductionmentioning

confidence: 99%

“…OUR has been extensively tested as the basis for an online softsensor and multiple methods to measure this parameter have been reported (Martínez-Monge et al, 2019b). Interesting OUR applications to monitor and control mammalian cell cultures include the work done by Europa et al (2000), who observed linearity between the OUR and the glucose consumption profile.…”

Section: Introductionmentioning

confidence: 99%

“…As a result, suboptimal titers and productivities are obtained, resulting in promising clones being discarded from scale‐up and further studies. Moreover, the metabolism of CHO cells is not constant throughout the culture process, but it shifts along with the experiment, making it even harder for the system to control substrate concentrations at optimal values (Martínez‐Monge et al, 2018; Zagari et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

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Soft‐sensors application for automated feeding control in high‐throughput mammalian cell cultures

Martínez-Monge

Martínez

Decker

et al. 2022

Biotech & Bioengineering

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The ever‐increasing demand for biopharmaceuticals has created the need for improving the overall productivity of culture processes. One such operational concept that is considered is fed‐batch operations as opposed to batch operations. However, optimal fed‐batch operations require complete knowledge of the cell culture to optimize the culture conditions and the nutrients feeding. For example, when using high‐throughput small‐scale bioreactors to test multiple clones that do not behave the same, depletion or overfeeding of some key components can occur if the feeding strategy is not individually optimized. Over the recent years, various solutions for real‐time measuring of the main cell culture metabolites have been proposed. Still, the complexity in the implementation of these techniques has limited their use. Soft‐sensors present an opportunity to overcome these limitations by indirectly estimating these variables in real‐time. This manuscript details the development of a new soft‐sensor‐based fed‐batch strategy to maintain substrate concentration (glucose and glutamine) at optimal levels in small‐scale multiparallel Chinese Hamster Ovary Cells cultures. Two alternatives to the standard feeding strategy were tested: an OUR soft‐sensor‐based strategy for glucose and glutamine (Strategy 1) and a dual OUR for glutamine and CO2/alkali addition for glucose soft‐sensor strategy (Strategy 2). The results demonstrated the applicability of the OUR soft‐sensor‐based strategy to optimize glucose and glutamine feedings, which yielded a 21% increase in final viable cell density (VCD) and a 31% in erythropoietin titer compared with the reference one. However, CO2/alkali addition soft‐sensor suffered from insufficient data to relate alkali addition with glucose consumption. As a result, the culture was overfed with glucose resulting in a 4% increase on final VCD, but a 9% decrease in final titer compared with the Reference Strategy.

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“…Os PCs podem ser absorvidos por alguns peptídeos e ter o transporte facilitado para a membrana plasmática. [14][15][16] As células HEK293, linha celular do rim embrionário humano, são utilizadas devido a sua alta reprodutibilidade, facilidade de cultivo e transfecção, sendo utilizadas na produção de proteínas e drogas e, por isso, são largamente utilizadas em estudos científicos [17]. Assim, este trabalho teve como objetivo sintetizar PCs por duas rotas distintas, utilizando ovalbumina como precursor, para utilizá-los como sondas fluorescentes para marcação de células HEK293.…”

Section: Introductionunclassified

Pontos de carbono luminescentes à base de ovalbumina aplicados em marcação biológica

et al. 2021

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RESUMO Entre os nanomateriais de carbono, os pontos de carbono (PCs) estão entre os mais atraentes devido as suas interessantes propriedades fotoluminescentes. Os PCs apresentam processo de síntese fácil, possibilidade de funcionalização da superfície, baixa toxicidade, alta solubilidade em água e biocompatibilidade. Devido a isso, suas aplicações na área biomédica e de bioimagem tem se destacado na literatura. Neste trabalho, PCs foram sintetizados por dois métodos diferentes a partir da ovalbumina, o qual é um precursor não tóxico e composto predominantemente por aminoácidos de cadeia curta. Os PCs foram caracterizados por espectroscopias de absorção (UV-Vis), de emissão (fluorescência) e infravermelho (IVTF), além de análise termogravimétrica (ATG). Os PCs foram utilizados como sondas fluorescentes para uma imagem colorida (azul e verde) de células HEK293 e não induziram a morte celular, o que indica que os mesmos são biocompatíveis e não tóxicos para este tipo de células. Dessa forma, os resultados aqui apresentados demonstram que os PCs podem ser considerados potenciais substitutos para corantes comerciais em marcação biológica.

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A new strategy for fed-batch process control of HEK293 cell cultures based on alkali buffer addition monitoring: comparison with O.U.R. dynamic method

Cited by 17 publications

References 37 publications

The Effect of the Expression of the Antiapoptotic BHRF1 Gene on the Metabolic Behavior of a Hybridoma Cell Line

The Effect of the Expression of the Antiapoptotic BHRF1 Gene on the Metabolic Behavior of a Hybridoma Cell Line

Soft‐sensors application for automated feeding control in high‐throughput mammalian cell cultures

Pontos de carbono luminescentes à base de ovalbumina aplicados em marcação biológica

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