Cell cycle phase dependent productivity of a recombinant Chinese hamster ovary cell line

Dutton, Roshni L.; Scharer, Jeno M.; Moo-Young, Murray

doi:10.1007/s10616-006-9041-4

Cited by 32 publications

(19 citation statements)

References 51 publications

(59 reference statements)

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“…S and G 2 /M) to the total productivity. A similar outcome is observed if the productivity is evaluated in terms of the cumulative cell hours [68] and the analysis is broken down per cell cycle phase [10]. Both a partially growth associated productivity and a better correlation towards the G 1 /G 0 phase is observed.…”

Section: Discussionsupporting

confidence: 67%

Cyclin and DNA Distributed Cell Cycle Model for GS-NS0 Cells

et al. 2015

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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 transition—have 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 cycle-specific 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.

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Section: Discussionsupporting

confidence: 67%

Cyclin and DNA Distributed Cell Cycle Model for GS-NS0 Cells

et al. 2015

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“…Depletion of glutathione with BSO has been shown to arrest cell division, suggesting that de novo glutathione synthesis is required for progression through the cell cycle and results in increased cell size . It has been reported that in CHO‐K1‐15 500 permanently producing tPA, cell size is a major determinant of productivity . However, this observation was not replicated in clones derived from a CHOK1SV pool transiently expressing cB72.3 .…”

Section: Discussionmentioning

confidence: 99%

Methionine sulfoximine supplementation enhances productivity in GS–CHOK1SV cell lines through glutathione biosynthesis

Feary

Racher

Young

et al. 2016

Biotechnology Progress

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In Lonza Biologics' GS Gene Expression System™, recombinant protein-producing GS-CHOK1SV cell lines are generated by transfection with an expression vector encoding both GS and the protein product genes followed by selection in MSX and glutamine-free medium. MSX is required to inhibit endogenous CHOK1SV GS, and in effect create a glutamine auxotrophy in the host that can be complemented by the expression vector encoded GS in selected cell lines. However, MSX is not a specific inhibitor of GS as it also inhibits the activity of GCL (a key enzyme in the glutathione biosynthesis pathway) to a similar extent. Glutathione species (GSH and GSSG) have been shown to provide both oxidizing and reducing equivalents to ER-resident oxidoreductases, raising the possibility that selection for transfectants with increased GCL expression could result in the isolation of GS-CHOKISV cell lines with improved capacity for recombinant protein production. In this study we have begun to address the relationship between MSX supplementation, the amount of intracellular GCL subunit and mAb production from a panel of GS-CHOK1SV cell lines. We then evaluated the influence of reduced GCL activity on batch culture of an industrially relevant mAb-producing GS-CHOK1SV cell line. To the best of our knowledge, this paper describes for the first time the change in expression of GCL subunits and recombinant mAb production in these cell lines with the degree of MSX supplementation in routine subculture. Our data also shows that partial inhibition of GCL activity in medium containing 75 µM MSX increases mAb productivity, and its more specific inhibitor BSO used at a concentration of 80 µM in medium increases the specific rate of mAb production eight-fold and the concentration in harvest medium by two-fold. These findings support a link between the inhibition of glutathione biosynthesis and recombinant protein production in industrially relevant systems and provide a process-driven method for increasing mAb productivity from GS-CHOK1SV cell lines. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:17-25, 2017.

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“…Further, product data plotted against the respective cumulative volumetric cell hours allowed for the characterization of protein production kinetics [11]. For example, Fig.…”

Section: Resultsmentioning

confidence: 99%

Statistical methods in media optimization for batch and fed-batch animal cell culture

Alwis

Dutton

Scharer

et al. 2007

Bioprocess Biosyst Eng

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Hybridoma 130-8F producing anti-F monoclonal antibodies (MAb) were grown in batch and fed-batch mode with glutamine as the limiting substrate. The initial concentration of glucose varied between 10 and 25 mM but was not growth limiting. Monoclonal antibody production was identified as being partially growth associated. Employing the cumulative cell hour concept, external metabolic flux estimates were calculated during the exponential growth phase for MAb, glucose, amino acids, ammonia and lactate. Through nutritional profiling using principal component analysis (PCA) followed by partial least squares regression (PLS), key metabolites were identified and grouped for significant positive, significant negative, low level, and negligible correlation to MAb production, cellular growth, glucose consumption, and ammonia and lactate production. Significant relationships peculiar to Hybridoma 130-8F were identified, such as demand for two normally non-essential amino acids (asparagine and aspartic acid), and the positive correlation between MAb and ammonia production.

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Cell cycle phase dependent productivity of a recombinant Chinese hamster ovary cell line

Cited by 32 publications

References 51 publications

Cyclin and DNA Distributed Cell Cycle Model for GS-NS0 Cells

Cyclin and DNA Distributed Cell Cycle Model for GS-NS0 Cells

Methionine sulfoximine supplementation enhances productivity in GS–CHOK1SV cell lines through glutathione biosynthesis

Statistical methods in media optimization for batch and fed-batch animal cell culture

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