On metabolic shift to lactate consumption in fed-batch culture of mammalian cells

Mulukutla, Bhanu Chandra; Gramer, Michael J.; Hu, Wei Shou

doi:10.1016/j.ymben.2011.12.006

Cited by 143 publications

(198 citation statements)

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“…In other words, the rate per cell of accumulation and consumption of L-lactic acid of CHO-M1 cells was faster than that of CHO-DP12 cells. It has been reported that lactate consumption in cell culture correlates with enhanced product yields in fed batch culture (Mulukutla et al 2012). In a survey of data from [200 manufacturing runs, a strong positive correlation between superior productivity and the switch from lactate production to consumption in late culture was observed.…”

Section: Metabolism Of Glucose and Glutamine In The Repeated Batch Cumentioning

confidence: 95%

Establishment of a mammalian cell line suitable for industrial production of recombinant protein using mutations induced by high-energy beam radiation

2013

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Mammalian cells are extensively used for production of biopharmaceuticals. Most cells used in industry have infinite proliferative capacity, which provides a high number of cells and corresponding productivity. However, infinite cells will continue to multiply even after cell density reaches sufficient levels. This excess proliferation aggravates the culture environment and induces low productivity. Therefore, after cell density reaches sufficient levels, downregulation of proliferation would prevent such aggravation and extend the culture period and improve productivity. To realize such suitable proliferation, we aimed to establish a novel cell line whose proliferation was spontaneously downregulated after reaching a sufficient population level. Mutagenesis using high-energy beam irradiation was used. CHO-DP12 cells were irradiated with 2.5 Gy X-rays and screened with hydroxyurea and 5-fluorouracil to eliminate any cells multiplying after confluence and to concentrate desired mutants. One clone was established and named CHO-M1. Cell cycle analysis indicated that CHO-M1 cells had a similar cell cycle profile in the exponential growth phase, but cells rapidly accumulated in G1 phase just before confluence and did not progress through the cell cycle. This suggested that until confluence, proliferation of CHO-M1 was similar to parental CHO, but after confluence, it was inhibited and under G1 arrest. The specific antibody production rate of CHO-M1 was kept high, even after confluence, while that of parental CHO was drastically decreased in stationary phase. These results suggest that the desired cell line was successfully established and that high-energy beam irradiation could be an efficient mutagenic technique for breeding industrial cells.

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Section: Metabolism Of Glucose and Glutamine In The Repeated Batch Cumentioning

confidence: 95%

Establishment of a mammalian cell line suitable for industrial production of recombinant protein using mutations induced by high-energy beam radiation

2013

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“…The adopted metabolic link herein is supported by studies that report a high conversion of glutamine into lactate [74] and the accumulation of glutamate when lactate is consumed [75]. Similarly, lactate consumption has been correlated to the cessation of rapid growth [55], which occurs in batch mode after the exhaustion of key substrates (such as glutamate) [50]. However, lactate consumption has been shown to be independent of the residual glucose and lactate concentrations in industrial fed-batch cultures [76].…”

Section: Discussionmentioning

confidence: 78%

“…In addition, lactate is also consumed (Q Lac ) [14,55], which in this case was modelled and triggered by the depletion of glutamate. Antibody production (q i , Equation 17) is specific for each cell cycle phase.…”

Section: Cyclins Growth Functionsmentioning

confidence: 99%

“…This dependency was introduced since it has been reported that glutamine, or glutamate in the GS system [50], is the main energy-yielding metabolic route [11,51,52] with over 90% flowing into the TCA cycle [53]. In addition, the cyclin growth functions (r i = G1,G2 ) were also formulated as lactate-dependent since it has been reported [54,55] that its concentration inhibits cell growth. Lactate dependency was not included in the DNA growth function (r i = S ), since it has been reported to be fairly constant in duration [56].…”

Section: Introductionmentioning

confidence: 99%

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A Cyclin Distributed Cell Cycle Model in GS-NS0

Munzer

Kostoglou

Georgiadis

et al. 2014

Computer Aided Chemical Engineering

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Mammalian cell cultures are intrinsically heterogeneous at different scales (molecular to bioreactor). The cell cycle is at the centre of capturing heterogeneity since it plays a critical role in the growth, death, and productivity of mammalian cell cultures. Current cell cycle models use biological variables (mass/volume/age) that are non-mechanistic, and difficult to experimentally determine, to describe cell cycle transition and capture culture heterogeneity. To address this problem, cyclins-key molecules that regulate cell cycle transitionhave been utilized. Herein, a novel integrated experimental-modelling platform is presented whereby experimental quantification of key cell cycle metrics (cell cycle timings, cell cycle fractions, and cyclin expression determined by flow cytometry) is used to develop a cyclin and DNA distributed model for the industrially relevant cell line, GS-NS0. Cyclins/DNA synthesis rates were linked to stimulatory/inhibitory factors in the culture medium, which ultimately affect cell growth. Cell antibody productivity was characterized using cell cyclespecific production rates. The solution method delivered fast computational time that renders the model's use suitable for model-based applications. Model structure was studied by global sensitivity analysis (GSA), which identified parameters with a significant effect on the model output, followed by re-estimation of its significant parameters from a control set of batch experiments. A good model fit to the experimental data, both at the cell cycle and viable cell density levels, was observed. The cell population heterogeneity of disturbed (after cell arrest) and undisturbed cell growth was captured proving the versatility of the modelling approach. Cell cycle models able to capture population heterogeneity facilitate in depth understanding of these complex systems and enable systematic formulation of culture strategies to improve growth and productivity. It is envisaged that this modelling approach will pave the model-based development of industrial cell lines and clinical studies.PLOS Computational Biology |

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“…This is evident from the data for 50 ml bioreactor which showed an increase in MAb production (B-D1 546 lg/ml, BFB-D1: 841 lg/ml) and a reduction in lactate production (B-D1: 24 mM/l, BFB-D1: 17 mM/l), a sign of slowing down of growth. The model simulation indicated that a high level of lactate can contribute to the reduction of glycolytic process (hence growth), as well as, acting as a driving force for its conversion to pyruvate (Mullkutla et al 2012). It is evident from the growth results for B-D1 and BFB-D1 (Fig.…”

Section: Verhulst Modelsmentioning

confidence: 83%

Revisiting Verhulst and Monod models: analysis of batch and fed-batch cultures

et al. 2014

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The paper re-evaluates Verhulst and Monod models. It has been claimed that standard logistic equation cannot describe the decline phase of mammalian cells in batch and fed-batch cultures and in some cases it fails to fit somatic growth data. In the present work Verhulst, population-based mechanistic growth model was revisited to describe successfully viable cell density (VCD) in exponential and decline phases of batch and fed-batch cultures of three different CHO cell lines. Verhulst model constants, K, carrying capacity (VCD/ml or lg/ml) and r, intrinsic growth factor (h -1 ) have physical meaning and they are of biological significance. These two parameters together define the course of growth and productivity and therefore, they are valuable in optimisation of culture media, developing feeding strategies and selection of cell lines for productivity. The Verhulst growth model approach was extended to develop productivity models for batch and fed-batch cultures. All Verhulst models were validated against blind data (R 2 [ 0.95). Critical examination of theoretical approaches concluded that Monod parameters have no physical meaning. Monodhybrid (pseudo-mechanistic) batch models were validated against specific growth rates of respective bolus and continuous fed-batch cultures (R 2 & 0.90). Thedescribes specific growth rate during metabolic shift (R 2 & 0.95). Verhulst substrate-based growth models compared favourably with Monod-hybrid models. Thus, experimental evidence implies that the constants in the Monod-hybrid model may not have physical meaning but they behave similarly to the biological constants in Michaelis-Menten enzyme kinetics, the basis of the Monod growth model.List of symbols K Carrying capacity for VCD K V-PB Carrying capacity, Verhust-population based for VCD K V-SB Carrying capacity, Verhust-substrate eased for VCD K V-MAb Carrying capacity, Verhust for MAb r Intrinsic growth factor r X Intrinsic productivity factor r V-PB Intrinsic growth factor, Verhulst population based for VCD r V-SB Intrinsic growth factor, Verhulst substrate based for VCD r V-MAb

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On metabolic shift to lactate consumption in fed-batch culture of mammalian cells

Cited by 143 publications

References 56 publications

Establishment of a mammalian cell line suitable for industrial production of recombinant protein using mutations induced by high-energy beam radiation

Establishment of a mammalian cell line suitable for industrial production of recombinant protein using mutations induced by high-energy beam radiation

A Cyclin Distributed Cell Cycle Model in GS-NS0

Revisiting Verhulst and Monod models: analysis of batch and fed-batch cultures

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