Patent ID: 7939296

Claim:
A method for production of recombinant human follicle-stimulating hormone (FSH) comprising (a) transfecting Chinese hamster ovary (CHO) cells with a first expression vector comprising a polynucleotide encoding the α-subunit of human FSH and a second expression vector comprising a polynucleotide encoding the β-subunit of human FSH, wherein said first and said second polynucleotides are under the regulation of an EF-1α promoter; (b) culturing said cells of (a) in a serum-free medium under conditions sufficient for the production of recombinant human FSH; (c) collecting culture supernatant by removing the cells from the culture that is obtained in step (b); (d) subjecting the culture supernatant collected in step (c) to cation-exchange column chromatography, wherein the cation exchanger employed in said cation-exchange column chromatography is a weak cation exchanger having a selectivity based on both hydrophobic interaction and hydrogen formation, and wherein the culture supernatant is loaded on the column and washed with a phosphate buffer of pH 5.0-6.5 supplemented with 50-250 mM of a salt; (e) eluting the recombinant human FSH from the column by increasing the concentration of said salt in said buffer to 300-900 mM to obtain one or more recombinant human FSH fractions; (f) subjecting the fractions collected in step (e) to dye affinity column chromatography to collect recombinant human FSH-active fractions, wherein the column used for the dye affinity column chromatography is a triazine dye affinity column, and wherein the fractions collected in step (e) are adjusted to or near the neutral pH with a buffer prior to application to the triazine dye affinity column equilibrated with a buffer at or near neutral pH; (g) eluting recombinant human FSH from the triazine dye affinity column of step (f) with a buffer having an elevated salt concentration of 1.8-2.2 mol/L; (h) subjecting the fractions collected in step (g) to hydrophobic column chromatography to collect recombinant human FSH-active fractions, wherein the column used for the hydrophobic column chromatography is a Phenyl-Sepharose column adjusted to or near the neutral pH with a buffer supplemented with 2-3 mol/L of a salt, and wherein the fractions collected in step (g) are adjusted to a salt concentration of 2.5-3.5 mol/L prior to application to the Phenyl-Sepharose column; (i) eluting the recombinant human FSH from the phenyl-Sepharose column by decreasing the concentration of said salt; and (j) subjecting the fractions collected in step (i) to gel filtration column chromatography to collect recombinant human FSH-active fractions.