FORTIFICATION OF a PROTEIN-FREE CELL CULTURE MEDIUM WITH PLANT PEPTONES IMPROVES CULTIVATION AND PRODUCTIVITY OF AN INTERFERON-γ–PRODUCING CHO CELL LINE

Abstract

A strong tendency is currently emerging to remove not only serum but also any product of animal origin from animal cell culture media during production of recombinant proteins. This should facilitate downstream processing and improve biosafety. One way consists in the fortification of protein-free nutritive media with plant protein hydrolysates. To investigate the effects of plant peptones on mammalian cell cultivation and productivity, CHO 320 cells, a clone of CHO K1 cells genetically modified to secrete human interferon-gamma (IFN-gamma), were first adapted to cultivation in suspension in a protein-free medium. Both cell growth and IFN-gamma secretion were found to be equivalent to those reached in serum-containing medium. Eight plant peptones, selected on the basis of their content in free amino acids and oligopeptides, as well as molecular weight distribution of oligopeptides, were tested for their ability to improve culture parameters. These were improved in the presence of three peptones, all having an important fraction of oligopeptides ranging from 1 to 10 kDa and a small proportion of peptides higher than 10 kDa. These peptones do not seem to add significantly to the nutritive potential to basal protein-free nutritive medium. Nevertheless, supplementation of an oligopeptide-enriched wheat peptone improved cell growth by up to 30% and IFN-gamma production by up to 60% in shake-flask experiments. These results suggest that the use of plant peptones with potential growth factor-like or antiapoptotic bioactivities could improve mammalian cell cultivation in protein-free media while increasing the product biosafety.

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

“…YP.A displayed concentrations of 5.1 9 10 6 cell mL -1 and 4.7 9 10 6 cell mL -1 , respectively, after only 90 h. During the first phase of culture (0-45 h) cells grew at a similar maximal specific rate of 0.04 h -1 whatever culture medium (Table 1). Several studies confirmed that maximal specific growth rates of CHO cells cultivated in Erlenmeyer flask were not enhanced by addition of YE or plant peptones (Sung et al 2004;Burteau et al 2003;Kim and Lee 2009). However, in the present study, the specific cell growth rate in control culture decreased from 0.04 h -1 to 0.01 h -1 during the phase from 45 to 80 h, while hydrolysates sustained its maximal value until 80 h of culture (Table 1).…”

“…Nevertheless, the risk of contamination by viruses, mycoplasma or prions from animal supplement led to a strong demand for media formulations free from animal components (Grillberger et al 2009). Then, the search for culture media that most closely replicated the benefits of animal-derived sera without their biosafety risks provoked a growing interest in the use of plant peptones (Heidemann et al 2000;Burteau et al 2003;Farges et al 2008) or yeast extracts (YE) (Sung et al 2004;Kim and Lee 2009).…”

“…On one hand, some results suggested the presence of growth factor-like and anti-apoptotic peptides from plant peptones (Burteau et al 2003;Farges et al 2008;Franek and Katinger 2002) or YE (Sung et al 2004), but without experimental validations. On the other hand, other studies concluded on nutritional efficiency of plant peptones as amino acid sources (Heidemann et al 2000;Nyberg et al 1999).…”

“…Metabolic flux analyses showed that a peptone obtained from an animal source promoted the entry of pyruvate to TCA cycle (Bonarius et al 1996). Other studies underlined that peptones improved cell growth without increasing carbon source consumption and lactate production (Ballez et al 2004;Chun et al 2007), while ammonia production was slightly increased (Burteau et al 2003). Moreover, some authors underlined the potential use of lactate as a source of carbon after glucose depletion, triggered by plant hydrolysates (Burteau et al 2003).…”

“…Other studies underlined that peptones improved cell growth without increasing carbon source consumption and lactate production (Ballez et al 2004;Chun et al 2007), while ammonia production was slightly increased (Burteau et al 2003). Moreover, some authors underlined the potential use of lactate as a source of carbon after glucose depletion, triggered by plant hydrolysates (Burteau et al 2003). These observations suggested that peptones could influence the cell metabolism depending on the culture phase.…”