Insulin Insight

Optimization of CHO-S cell culture medium by supplementation with non-animal derived components using DoE

Optimization of CHO-S cell culture medium by supplementation with non-animal derived components using DoE

Author: Francesc Gòdia Casablancas, Universitat Autònoma de Barcelona

The main objective of the study was to investigate the effect of insulin as supplement to improve cell culture media for the growth of a CHO cell line. The approach of the study was similar to a previous work of UAB on the optimization of commercial serum free, chemically defined and animal derived component free media by supplementation with recombinant compounds, insulin among them, performed on HEK 293 cells. Nowadays, CHO are one of the most widely used platforms for the production of biopharmaceuticals. The cell line CHO-S was selected to perform the study.

The use of serum-free medium has grown significantly in industrial applications where the use of bovine serum represents a safety hazard as well as a source of unwanted contamination for the production of biopharmaceuticals. Serum-free medium is prepared without the use of animal serum, but may contain serum constituents or substitutes thereof.

In this work, three commercial serum-free and chemically defined media were initially trade-off, and one of them (FreeStyleCHO) was selected to perform a further optimization based on testing the effect of eight non animal-derived compounds as supplements (r-insulin, r-transferrin, r-albumin, selenium, tocopherol acetate, synthetic cholesterol, tween 80 and fatty acids) in order to increase maximum cell density. Design of experiments methodologies was used to minimize the number of experiments, elucidate cross effects between compounds, as well as to evaluate the statistical significance of the obtained results.

Plackett-Burman initial screening experiments indicated that only three of the supplements (r-insulin, r-transferrin and selenium) had positive effects on cell growth. The concentration of these components was further optimized by means of a Box-Behnken design. By using this strategy, a maximum cell density of 10 x 106 cells/mL was attained, a high cell concentration for batch culture operation, with FreeStyleCHO medium supplemented with selenium (1 µg/L), r-transferrin (20.2 mg/L) and r-insulin (26.1 mg/L).

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