Strategies for fed-batch cultivation of t-PA producing CHO cells: substitution of glucose and glutamine and rational design of culture medium.
A strategy for fed-batch cultivation of t-PA producing recombinant CHO cells is presented, based on the substitution of glucose and glutamine for slowly metabolized nutrients and in a rational design of the medium. Media for the batch and fed stages were based on the cell specific amino acid requirements, which allowed a more accurate determination of the initiation of the fed stage and the frequency of nutrient addition from then on. Salt concentration was also reduced in both media to avoid an increase in osmolality. As a consequence of this rational design, most amino acid did not accumulate significantly during the fed stage, as usually occurs when their supply is not based on cell requirements; also, lower amounts of by-products were obtained when osmolality level was kept low, that altogether increased viability, longevity and t-PA production when compared with a reference batch culture. Alternating glucose and galactose during the fed stage, allowed lactate detoxification of the cells through their own metabolism. This allowed an increase in cell growth and cell viability with respect to a fed-batch culture in which only glucose was used in the fed stage.
Altamirano
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Escuela de Ingeniería Bioquímica, Facultad de Ingeniería, Universidad Católica de Valparaíso, Av. Brasil 2147, Valparaíso, Chile. claudia.altamirano@ucv.cl
Paredes
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Illanes
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Cairó
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Gòdia
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D000818:Animals; D016466:CHO Cells; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media; D005690:Galactose / Q000032:analysis / Q000378:metabolism; D005947:Glucose / Q000032:analysis / Q000378:metabolism*; D018698:Glutamic Acid / Q000032:analysis / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism*; D019344:Lactic Acid / Q000032:analysis / Q000378:metabolism; D013997:Time Factors; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis*
D003470:Culture Media; D005973:Glutamine; D019344:Lactic Acid; D018698:Glutamic Acid; D010959:Tissue Plasminogen Activator; D005947:Glucose; D005690:Galactose