Peptone Supplementation of Culture Medium Has Variable Effects on the Productivity of CHO Cells

Abstract

The optimization of cell culture conditions for growth and productivity of recombinant Chinese hamster ovary (CHO) cells is a critical step in biopharmaceutical manufacturing. In the present study, the effects of the timing and amount of peptone feeding of a recombinant CHO cell line grown in a basal medium in serum-free suspension culture were determined for eight peptones of different origin (plant and casein). The amino acid content and the average molecular weight of the peptones chosen were available. In optimized feeding strategies with single peptones, increase 100 % volumetric productivity and 40 % in cell number were achieved. In feeding strategies with two peptones, several combinations stimulated protein productivity more than either peptone alone, depending on the peptone concentration and time of feeding. Some peptones, which did not stimulate productivity when added alone proved to be effective when used in combination. The combined peptones feeding strategies were more effective with peptones of different origin. Our data support the notion that the origin of peptones provides some guidance in identifying the most effective feeding strategies for recombinant CHO cells.

Keywords: Feeding strategy; mammalian cell culture; plant peptones; recombinant proteins; stable CHO cell lines.

Fig. 1

The effect of peptones on cell density, biomass and PCV values in different media. Panel a: CHO clone 1 cells were inoculated in RPMI 1640 medium and the different peptones were added at a concentration of 2 g /L on two different times: the day of inoculation (day 0, ■) or 2 days after inoculation (day 2, ■). The relative biomass increase of cultures supplemented with peptones was expressed as a percentage (%) increase to the PCV value of parallel cultures without peptones. Data were measured on day 3 of culture. Panel b: Viable cell number (columns) and viability (dots) of clone 1 cultivated in RPMI 1640 medium in the presence of 2 g /L of the indicated peptones, measured on day 3. The indicated peptones were added at the time of cell inoculation. Panel c: PCV values (%) of clone 1 cultivated in RPMI 1640 medium in the presence of 2 g /L of the indicated peptones and measured on day 3. The indicated peptones were added at the time of cell inoculation. Panel d: PCV values (%) of clone 1 cultivated in LBTC-CDM medium in the presence of 2 g /L of the indicated peptones and measured on day 3. The indicated peptones were added at the time of cell inoculation

Fig. 2

Variation in IgG production levels in the presence of single peptones. CHO clone 1 was cultivated in LBTC-CDM medium in the presence of the indicated peptone at 2 g /L or 4 g /L added on day 0, day 2, or day 4 of culture. Data are expressed as the percentage increase in titers relative to the control culture without peptone. The control cultures produced between 60 and 80 mg IgG /L after 7 days. Cells were inoculated at a density of 1 x 10⁶ cells mL

Fig. 3

Effect of mixed peptone feeding at 2 g/L on IgG production. CHO clone 1 cells were cultivated in LBTC-CDM medium in the presence of 1:1 (w/w) mixtures of 2 peptones at 1 g/L final concentration of each peptone (total peptone concentration: 2 g/L) added on day 0 (■) or day 2 (■) post-inoculation. Peptones mixes are indicated above the x-axis. Data are expressed as a percentage variation in IgG yield relative to the control culture without peptones. The yield of the control cultures was between 120 and 180 mg IgG/L after 7 days. Cells were inoculated at a density of 1 x 10⁶ cells mL

Fig. 4

Effect of mixed peptone feeding at 4 g/L on IgG production. To cultures of CHO clone 1 cells in LBTC-CDM medium, mixes of two peptones were added at 2 g/L final concentration of each peptone (total concentration: 4 g peptone/L) on 3 different cultivation days (day 0, day 2, or day 4). Peptones mixes are indicated above the x-axis. The data are expressed as the percentage variation in IgG production relative to the control cultures without peptones. The control yielded between 120 and 180 mg IgG/L after 7 days of culture. Cells were inoculated at a density of 1 x 10⁶ cells mL.

Fig. 5.

Effect of mixed peptone feeding strategies compared to single peptone feeding strategies.Data on the Y-axis value express the difference in productivity expressed as fold variation over control without peptone, calculated according to the formula: Y = V_(a+b) – [(V_a + V_b)/2] where: V_(a+b) represents the fold variation in productivity of the 2-peptone mix at 4 g/L (data from Fig. 4); V_a and V_b represent the fold variation in productivity of the single peptones (a) and (b) added each at 2 g/L (data from Fig.2); Consequently, [(Va + Vb)/2] represents the expected average effect of the two individual peptones. The data obtained were grouped by peptone category and by day of peptone addition post-inoculation (0 = day 0, black dots; 2 = day 2, grey dots). Categories on the X-axis: C, casein + casein; S, soy + soy; CW, casein + wheat; CS, casein + soy; WS, wheat + soy. The numbers refer to the day of peptone addition. A zero value represents no difference in productivity to the expected average effect of single peptone addition