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

Abstract

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.

Fig. 1

Composition of yeast hydrolysates: A carbohydrates (open bar), nucleic acids (vertical lines filled bar), peptides (bar with upper left to lower right fill) and free amino acids (filled bar) in yeast extract (YE), peptone A (YP.A) and peptone B (YP.B). B Molecular size distribution of peptides from YE (red colour solid line), YP.A (blue colour dotted line) and YP.B (dashed line)

Fig. 2

Maximal cell concentration (A) and IgG production (B) of CHO-AMW cells cultivated in Erlenmeyer flask: without supplementation (horizontal lines filled bar), with supplementation of 1 g L⁻¹ (open bar) or 4 g L⁻¹ (filled bar) of YE, YP.A and YP.B. Maximal cell concentration (C) and IgG production (D) of CHO-AMW cells cultivated in Erlenmeyer flask: without supplementation (horizontal lines filled bar), with supplementation of mixture of 1 g L⁻¹ YE and 4 g L⁻¹ YP.A or YP.B at the seeding time (open bar) or after 40 h (filled bar). Control was performed without yeast hydrolysates supplementation. Maximal concentrations of cell and IgG were measured after 90 h of culture. The impact of yeast hydrolysates conditions of supplementation on maximal concentration of cell and IgG was first verified thanks to an analysis of variance (ANOVA). Possible significant differences between hydrolysates conditions of supplementation on the maximal concentration of cells and IgG were evaluated by least significant differences (LSD) multiple comparison tests with a confidence level of 95 %. 7 control cultures were conducted independently, while yeast hydrolysates supplemented cultures were repeated three times. The letters represent similar groups

Fig. 3

Kinetics of CHO cells cultivated in Erlenmeyer flask (a–c) and in bioreactor (A–C): a, A viable cells; (b, B) Trypan blue dead cells (open symbols) and lysed cells (closed symbols); c, C IgG production, performed without supplementation (open circle), with 1 g L⁻¹ YE (open triangle), or with 4 g L⁻¹ YP.A + 1 g L⁻¹ YE (open square)

Fig. 4

Kinetics of CHO cells cultivated in Erlenmeyer flask (a, b) and bioreactor (A, B): a, A viable cells (open symbols) and late apoptotic cells (closed symbols); b, B early apoptotic cells, performed without supplementation (open circle), with 1 g L⁻¹ YE (open triangle), or with 4 g L⁻¹ YP.A +1 g L⁻¹ YE (open square)

Fig. 5

Kinetics of substrate consumption and metabolite production in Erlenmeyer flask (a, b and c) and bioreactor (A, B and C): a, A glucose consumption (open symbols) and lactate production (closed symbols); b, B correlation between produced lactate and consumed glucose; c, C correlation between produced cells and consumed glucose. Cultures performed without supplementation (open circle), with 1 g L⁻¹ YE (open triangle), or with 4 g L⁻¹ YP.A + 1 g L⁻¹ YE (open square). Arrows represent metabolism shift

Fig. 6

Kinetics of substrate consumption and metabolite production in Erlenmeyer flask (a, b) and bioreactor (A, B): a, A glutamine consumption (open symbols) and ammonia production (closed symbols); b, B Correlation between produced cells and consumed glutamine. Cultures performed without supplementation (open circle), with 1 g L⁻¹ YE (open triangle), or with 4 g L⁻¹ YP.A + 1 g L⁻¹ YE (open square). Arrows underline the use of another source of substrate that partially replaced glutamine