Decoupling cell growth and product formation in Chinese hamster ovary cells through metabolic control

Altamirano, Claudia; Cairó, Jordi J.; Gòdia, Francesc

doi:10.1002/bit.10096

Cited by 60 publications

(50 citation statements)

References 49 publications

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“…The apparent G1 association of tPA production from CHO cells observed in this study is in agreement with the work of Altamirano et al (2001) who observed tPA production from a growth-arrested CHO cell population. This is in contrast, however, with the findings of Fann et al (2000), who in an experimental design limited to investigation of the exponential population growth phase, found a direct correlation of tPA production from CHO cells to the specific growth rate, implying S cell cycle phase dependence.…”

Section: Dhfr Expressionsupporting

confidence: 93%

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

Dutton¹,

Scharer

Moo-Young

2007

Cytotechnology

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A Chinese Hamster Ovary cell line, CHO1-15 500 , producing recombinant human tissue type plasminogen activator (tPA) via the dihydrofolate reductase (DHFR) amplification system, was studied in batch culture. In this system both DHFR and tPA are under the control of the strong constitutive viral SV40 early promoter. Employing the cumulative viable cell-hour approach, the specific productivity of tPA had maxima in the lag (0.065 pg cell -1 h -1 ) and early decline (0.040 pg cell -1 h -1 ) population growth phases. The viable population was assigned into four subpopulations (G1, S, G2/M phase, and Apoptotic cells) using flow cytometric analysis. As expected, intracellular DHFR was maximally expressed during the S cell cycle phase. The production of tPA, however, was found to be a direct linear function of the G1 phase, with a subpopulation specific productivity of 0.080 pg c-h -1. Productivity maxima in the lag and early decline corroborate the flow cytometric data, indicative that this recombinant tPA production occurs primarily in the G1 cell cycle phase, not the S phase. This suggests that endogenous regulatory mechanisms are important controlling influences on the production of recombinant tPA in this ovarian cell line. Productivity from recombinant cell lines cannot be inferred from either the plasmid construct or the host cell alone. Elucidation of the relationship between expression of recombinant protein and the cell cycle phases of the host cell is a major component of the characterization of the animal cell production system. This information facilitates rational process design, including operating mode, modelling and control, and medium formulation.Keywords Cell cycle associated protein productivity Á Chinese hamster ovary (CHO) Á DHFR Á Regulation of expression Á Specific productivity Á Tissue plasminogen activator (tPA) Abbreviation CH vol volumetric cell-hours

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Section: Dhfr Expressionsupporting

confidence: 93%

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

Dutton¹,

Scharer

Moo-Young

2007

Cytotechnology

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“…Control of proliferation has also been pursued by a series of other research groups who described very different strategies of controlling the progress of cells throughout the cell cycle. Cell-cycle arrest in G 1 has been induced in several ways: by starvation of an essential nutrient (Seifert and Phillips, 1999), by the addition of DNA synthesis inhibitors (Al-Rubeai et al, 1992), by the addition of genotoxic agents (Gartenhaus et al, 1996), through metabolic control (Altamirano et al, 2001) and by over-expression of cell-cycle inhibitors (Kaufmann et al, 2001;Mazur et al, 1998). A simple and wellestablished method based on changes of process parameters is the concept of growth arrest by shifting the culture to low temperatures.…”

Section: Discussionmentioning

confidence: 99%

Process parameter shifting: Part II. Biphasic cultivation—A tool for enhancing the volumetric productivity of batch processes using Epo‐Fc expressing CHO cells

Trummer

Fauland

Seidinger

et al. 2006

Biotech & Bioengineering

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Regulation of cell growth and protein expression potentially results in a sustainable enhancement of the volumetric productivity in a fermentation process. Following a biphasic cultivation strategy the process initially passes through a cell proliferation phase to generate a sufficiently high viable cell mass. In the subsequent production phase cells are maintained viable and productive without significant cell proliferation leading to increased viable cell days and product yields. In a previous work we have shown that the well directed alteration of the process environment based on process parameter shifting is a promising tool to regulate cell growth and protein expression. In continuation of this work we investigated process parameters which have been identified to affect cell proliferation in favor of an increased specific productivity and total product yield in a series of biphasic batch cultivation experiments. In most of these processes the integral of viable cells and the specific productivity were increased leading to a significant improvement of both final product concentration and volumetric productivity. In addition, combined parameter shifts (pH 6.90/30 degrees C and pH 6.90/33 degrees C) exerted a synergistic effect on product quality. The loss of product sialylation which occurred at reduced temperatures was prevented by simultaneously reducing the external pH. In conclusion, biphasic cultivation based on combined shifting of process parameters is a suitable tool for controlling cell proliferation and protein expression of mammalian cells in a batch bioreactor leading to enhanced volumetric productivities and therefore offers an enormous potential for bioprocess optimization.

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“…The use of alternative sugar sources is a commonly used strategy to improve recombinant protein production in animal cell culture. Galactose, sorbitol, and fructose are slowly metabolized carbon sources that have been tested as alternatives to glucose to improve antibody production in hybridomas (Mochizuki et al, 1993;Petch and Butler, 1996) and tissue plasminogen activator in Chinese ovary hamster cells (CHO) (Altamirano et al, 2000(Altamirano et al, , 2001. Similarly, it was observed that the use of fructose and galactose in several retrovirus producer cell lines (Te Fly, PG13, Tel CeB) diminished the production of lactate and simultaneously vector titers were improved three to eightfold (Gény-Fiamma et al, 2003;Merten, 2004;Merten et al, 2001).…”

Section: Introductionmentioning

confidence: 87%

Effect of medium sugar source on the production of retroviral vectors for gene therapy

Coroadinha

Ribeiro

Roldão

et al. 2006

Biotech & Bioengineering

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The production of gene therapy retroviral vectors presents many difficulties, mainly due to vector instability and low cell productivities hampering the attainment of high titers of infectious viral vectors. The objective of this work is to increase the production titers of retroviral vectors by manipulating the sugar carbon sources used in bioreaction. Four sugars were tested (glucose, galactose, sorbitol, and fructose) on an established Moloney murine leukemia virus (MoMLV) producer cell line. Galactose and sorbitol did not support cell growth or vector production. Glucose supplemented at 25 mM supported the highest cell growth; however, the use of glucose or fructose at 83 and 140 mM have shown to improve the infectious vector titer three to fourfold. The reasons for the titer improvements were further analyzed and, although, the cell-specific productivity in viral transgene RNA and reverse transcriptase were augmented 5- and 6-fold for glucose at 140 mM and 14- and 16-fold for fructose at 140 mM, comparing with glucose at 25 mM, these increases did not seem sufficient to account for the 14- (140 mM glucose) and 32- (140 mM fructose) fold increment obtained for the infectious particles-specific productivity. Further accounting the enhancement in the titers was the improvement in the viral stability, the half-life of the vectors was enhanced by 30-60%. This resulted in a product quality with a superior ratio of infectious to total particles, thus reducing the most problematic contaminant in the production of retroviral vectors, non-infectious retroviral particles.

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Decoupling cell growth and product formation in Chinese hamster ovary cells through metabolic control

Cited by 60 publications

References 49 publications

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

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

Process parameter shifting: Part II. Biphasic cultivation—A tool for enhancing the volumetric productivity of batch processes using Epo‐Fc expressing CHO cells

Effect of medium sugar source on the production of retroviral vectors for gene therapy

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