Patent ID: 8609370

Claim:
A process for the production of a highly active sialylation form of a target glycoprotein, said highly active sialylation form of said target glycoprotein having a sialylation degree that is lower than that of a reference glycoprotein, yet said highly active sialylation form of said target glycoprotein also having an activity that is higher than that of said reference glycoprotein, wherein said sialylation degree also corresponds to a determined concentration of at least one sialic acid precursor additive, said process comprising: (a) providing a human expression cell line which harbors at least one defect in the sugar nucleotide biosynthetic pathway of sialic acids, said at least one defect being a defect in at least one enzyme selected from the group consisting of UDP-GlcNAc-2-epimerase, N-acetylmannosamine kinase, N-acetylglucosamine kinase, Neu5Ac-9-P-synthetase, Neu5Ac-9-P-phosphatase, CMP-Neu5Ac synthetase and CMP-sialic acid transporter, and said human expression cell line being transfected with a nucleic acid encoding the target glycoprotein; (b) determining a concentration of said at least one sialic acid precursor additive that is able to reconstitute said defect by a process comprising: (i) expressing a plurality of different sialylation forms of said target glycoprotein by differential sialylation using different concentrations of said at least one sialic acid precursor additive; (ii) determining the activity of each of the different sialylation forms in comparison to the reference glycoprotein; and (iii) selecting said highly active sialylation form of said target glycoprotein from among the different sialylation forms expressed, and determining the concentration of the at least one sialic acid precursor additive that correlates with the expression of said highly active sialylation form of said target glycoprotein; and (c) expressing the highly active sialylation form of said target glycoprotein in said human expression cell line in a medium supplemented with the at least one sialic acid precursor additive at the concentration determined in step (b)(iii).