Chromatographic fractionation of yeast extract: A strategy to identify physicochemical properties of compounds promoting CHO cell culture

Mosser, Mathilde; Kapel, Romain; Aymes, Arnaud; Bonanno, Laurent-Michel; Olmos, Éric; Chevalot, Isabelle; Marc, Ivan; Marc, Annie

doi:10.1016/j.procbio.2012.04.015

Cited by 7 publications

(14 citation statements)

References 29 publications

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“…The molecular size distribution of the peptides contained in yeast hydrolysates was set out by analytical size exclusion high performance liquid chromatography (SE-HPLC) using a Superdex peptide column coupled to an UV detector (Mosser et al 2012). …”

Section: Molecular Size Distributionmentioning

confidence: 99%

“…Amino acids were derivatized with 9-fluoroenylmethyl chloroformate and o-phthalaldehyde and analyzed by reverse phase HPLC according to the conditions previously described (Mosser et al 2012). The amino acid concentrations were calculated from calibration curves performed with an amino acid kit (Sigma-Aldrich Co., St. Louis, MO, USA).…”

Section: Total and Free Amino Acidsmentioning

confidence: 99%

“…Then, monosaccharides were analyzed by ion exchange HPLC according Control culture was performed without yeast hydrolysate supplementation Control culture was performed without yeast hydrolysate supplementation to the conditions previously described (Mosser et al 2012). The concentration of mannose and glucose, which were the main monosaccharides in yeast polysaccharides, was calculated from calibration prepared with standard solutions (Sigma-Aldrich Co.).…”

Section: Carbohydratesmentioning

confidence: 99%

“…The solutions were neutralized in NH 4 H 2 PO 4 (2 M) and filtered through a membrane of 0.22 lm pore size. Then, the nucleobases (adenine, cytosine, uracile, guanine, thymine and hypoxanthine) were analyzed by reverse phase HPLC according to the conditions previously described (Mosser et al 2012). The nucleobase concentrations were calculated from calibration performed with standards (SigmaAldrich Co.).…”

Section: Nucleic Acidsmentioning

confidence: 99%

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Combination of yeast hydrolysates to improve CHO cell growth and IgG production

et al. 2012

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Many studies underlined the great benefits of hydrolysates used as additives in animal free media on cell culture performances. However, to precisely define hydrolysate supplementation strategies, a deeper understanding of their effect on cell growth and protein production is required. In the present study, the effect of addition of one yeast extract (YE) and two yeast peptones (named YP.A and YP.B) in a chemically defined medium was first assessed on cell culture performances. Interestingly, specific effects were found depending on the degree of degradation of yeast hydrolysates. The YE at 1 g L -1 increased the maximal cell density by 70 %, while a mixture of YE (1 g L -1 ) and YP.A (4 g L -1 ) increased IgG production by 180 %. These conditions were then evaluated on the CHO cell kinetics all over cultures. Hydrolysates extended the cell growth phase in Erlenmeyer flask and increased the maximal growth rate in bioreactor up to 20 %. Cell growth stimulation induced by hydrolysates addition was linked with energetic metabolism improvement suggesting that they promote oxidative pathway. Furthermore, hydrolysates provided an additional source of substrate that supported cell growth despite glutamine limitation.

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Section: Molecular Size Distributionmentioning

confidence: 99%

Section: Total and Free Amino Acidsmentioning

confidence: 99%

Section: Carbohydratesmentioning

confidence: 99%

Section: Nucleic Acidsmentioning

confidence: 99%

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Combination of yeast hydrolysates to improve CHO cell growth and IgG production

et al. 2012

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“…While some reports attributed the improved cell growth and productivity to the nutritional enrichment by addition of hydrolysates (Nyberg et al 1999), some recent results proposed that Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-6789-5) contains supplementary material, which is available to authorized users. possible peptides in hydrolysates with growth-promoting, anti-apoptotic, or production-enhancing effects played the main role (Mosser et al 2012;Sung et al 2004). On the other hand, a kinetic study of Chinese hamster ovary (CHO) cells highlighted that cell growth phase extended by YE addition resulted from the TCA cycle supplement and improvement of oxidative metabolism (Mosser et al 2013).…”

Section: Introductionmentioning

confidence: 99%

Understanding the intracellular effects of yeast extract on the enhancement of Fc-fusion protein production in Chinese hamster ovary cell culture

Sun

Liu

et al. 2015

Appl Microbiol Biotechnol

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Yeast extract (YE), as a non-animal source additive for mammalian cell culture medium, has been widely used for manufacturing of therapeutic proteins. In the present study, one particular YE was found to have significantly improved the specific productivity (q p) of Fc-fusion protein in recombinant Chinese hamster ovary (rCHO) cell culture. In order to elucidate the intracellular effects of YE on protein productivity, steps of the target protein synthesis process were investigated to unveil their variations caused by YE addition. Stepwise analysis on Fc-fusion protein synthesis process showed that YE enhanced Fc-fusion protein gene transcription with cell cycle arrest at G1 phase; mammalian target of rapamycin (mTOR) signaling pathway was activated to enhance the translation of Fc-fusion protein, and the block in post-translational steps of Fc-fusion protein was alleviated by YE addition as well. Our results revealed the responses of multiple protein production steps to the addition of YE and provided a practical guidance for the separation and application of active compounds from hydrolysates.

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Fractionation of yeast extract by nanofiltration process to assess key compounds involved in CHO cell culture improvement

Mosser

Kapel

Chevalot

et al. 2015

Biotechnology Progress

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Yeast extract (YE) is known to greatly enhance mammalian cell culture performances, but its undefined composition decreases process reliability. Accordingly, in the present study, the nature of YE compounds involved in the improvement of recombinant CHO cell growth and IgG production was investigated. First, the benefits of YE were verified, revealing that it increased maximal concentrations of viable cells and IgG up to 73 and 60%, respectively compared to a reference culture. Then, the analyses of YE composition highlighted the presence of molecules such as amino acids, vitamins, salts, nucleobase, and glucose that were contained in reference medium, while others including peptides, trehalose, polysaccharides, and nucleic acids were not. Consequently, YE was fractionated by a nanofiltration process to deeper evaluate its effects on CHO cell cultures. The YE molecules already contained in reference medium were mainly isolated in the permeate fraction together with trehalose and short peptides, while other molecules were concentrated in the retentate. Permeate, which was free of macromolecules, exhibited a similar positive effect than raw YE on maximal concentrations. Additional studies on cell energetic metabolism underlined that dipeptides and tripeptides in permeate were used as an efficient source of nitrogenous substrates.

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Chromatographic fractionation of yeast extract: A strategy to identify physicochemical properties of compounds promoting CHO cell culture

Cited by 7 publications

References 29 publications

Combination of yeast hydrolysates to improve CHO cell growth and IgG production

Combination of yeast hydrolysates to improve CHO cell growth and IgG production

Understanding the intracellular effects of yeast extract on the enhancement of Fc-fusion protein production in Chinese hamster ovary cell culture

Fractionation of yeast extract by nanofiltration process to assess key compounds involved in CHO cell culture improvement

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