abstract |
The disclosure provides a method of producing recombinant alkaline phosphatase comprising: (i) inoculating Chinese Hamster Ovary (CHO) cells expressing recombinant alkaline phosphatase in culture medium; (ii) culturing the CHO cells in the culture medium at a temperature of about 37 °C; (iii) adding a combination of nutrient supplements to the cell culture of (ii) at least one day after inoculation, the combination comprising (a) a first animal-derived component-free (ADCF) nutrient supplement comprising one or more amino acids, vitamins, salts, trace elements, poloxamer and glucose, wherein the first ADCF nutrient supplement does not comprise hypoxanthine, thymidine, insulin, L-glutamine, growth factors, peptides, proteins, hydrolysates, phenol red and 2-mercaptoethanol; and (b) a second ADCF nutrient supplement comprising one or more amino acids, wherein the second ADCF nutrient supplement lacks hypoxanthine, thymidine, insulin, L-glutamine, growth factors, peptides, proteins, hydrolysates, phenol red, 2-mercaptoethanol and poloxamer; (iv) decreasing the temperature of the cell culture of (iii) to about 30 °C about 80 hours to 120 hours after the innoculation; and (v) isolating the recombinant alkaline phosphatase from the cell culture of (iv) by at least one chromatography step. |