Enhanced recombinant M-CSF production in CHO cells by glycerol addition: model and validation

Abstract

Addition of stimulatory chemical such as glycerol was found to increase recombinant protein production in Chinese hamster ovary (CHO) cells. However, glycerol influenced cell mitosis and reduced cell growth rate. We developed a controlled proliferation strategy to utilize the stimulation of glycerol on recombinant protein production and mitigate the problem of growth inhibition. The approach is to apply a two-stage process, where cells are cultured without glycerol for a period of time in order to obtain enough cell density and then glycerol is added to achieve high specific productivity. In addition, a model for predicting the profiles of cell proliferation and recombinant protein production was developed and validated. A two-stage process, addition of 1% glycerol after 1 day of growth, could increase the final production of macrophage-colony stimulating factor (M-CSF) by 38% compared with the value obtained without addition of glycerol.

Fig. 1

Effects of glycerol concentration on CHO cell proliferation and M-CSF production. The results are an average of two separate experiments (each in triplicate in 12-well tissue culture plates). Error bars in the figure stand for one standard deviation (SD)

Fig. 2

Time course of cell proliferation, M-CSF, and glucose consumption for CHO cells. Control culture and culture with 1% glycerol supplement at day 0 were cultivated to obtain these data. The results are an average of two separate experiments (each in triplicate in 12-well tissue culture plates) and the coefficient of variance is less than 10%

Fig. 3

Specific growth rate (a, b) and specific glucose consumption rate (c, d) as a function of glucose concentration. The specific growth rate and glucose consumption rate were obtained from data of Fig. 2. The glycerol was added at the concentration of 1%. The dashed lines were determined by using the hyperbola regression in the Sigmaplot package. The results are an average of two separate experiments (each in triplicate in 12-well tissue culture plates) and the coefficient of variance is less than 10%

Fig. 4

M-CSF production as a function of the integral of viable cell density (IVCD). Average specific production rate, q_M-CSF, is the slope of the least–squared linear fit for experimental data from cultures without glycerol (control) and with glycerol supplement. The glycerol was added at the concentration of 1%. The results are an average of two separate experiments (each in triplicate in 12-well tissue culture plates) and the coefficient of variance is less than 10%

Fig. 5

Profiles of cell proliferation and M-CSF production for cultures with 1% glycerol added at day 1, 2, 3, and 4. The model (Eqs. 1–3) in conjunction with kinetic parameters in Table 1 was used to predict the profiles of M-CSF production and cell proliferation with the aid of the built-in function “NDSolve” in the Mathematica package. Model prediction (dash line) compared with actual cell proliferation (closed circle) and actual M-CSF (open circle). The results are an average of two separate experiments (each in triplicate in 12-well tissue culture plates). Error bars in the figure stand for one standard deviation (SD)