Patent ID: 11913039
Assignee: ALEXION PHARMACEUTICALS, INC.
Field: Pharmaceuticals (Chemistry)
Classification: CPC C  A | IPC C

Claim 0:
1. A method of producing recombinant alkaline phosphatase comprising:
(I) (a) inoculating Chinese Hamster Ovary (CHO) cells expressing recombinant alkaline phosphatase;
(b) culturing the CHO cells in culture medium;
(c) isolating the recombinant alkaline phosphatase from the culture medium by at least one purification step to form harvest clarified culture fluid (HCCF) with a total sialic acid content (TSAC) of about 2.1 mol/mol to about 4.3 mol/mol;
(d) performing at least one additional protein purification step to form a filtration pool (UFDF), wherein the UFDF is held at a temperature of about 13° C. to about 27° C. for about 14 hours to about 42 hours, and at a protein concentration of about 2.0 g/L to about 4.3 g/L to reduce TSAC loss; and
(e) subjecting the UFDF to at least one chromatography step to obtain partially purified recombinant alkaline phosphatase; and
(f) recovering the recombinant alkaline phosphatase from the UFDF, wherein the recombinant alkaline phosphatase has a TSAC of about 1.2 mol/mol to about 3.0 mol/mol; or

(II) (a) inoculating CHO cells expressing recombinant alkaline phosphatase;
(b) culturing the CHO cells in culture medium to produce a cell culture;
(c) adding a nutrient supplement to the culture medium;
(d) isolating the recombinant alkaline phosphatase from the culture medium by at least one purification step to form a filtration pool (Ultrafiltration/Diafiltration UFDF), wherein the UFDF is held at a temperature of about 13° C. to about 27° C. for about 14 hours to about 42 hours, and at a protein concentration of about 2.0 g/L to about 4.3 g/L to reduce TSAC loss; and
(e) recovering the recombinant alkaline phosphatase from the UFDF, wherein the recombinant alkaline phosphatase in the UFDF has a TSAC of about 1.2 mol/mol to about 3.0 mol/mol.