Method of producing and purifying an active soluble sialyltransferase
AssigneeBiogenerix AG
InventorsAxel Angermann, Christian Scheckermann, Karsten Schmidt
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Abstract
The present invention relates to a method for the production and purification of a sialyltransferase polypeptide, in particular a N-Acetylgalactosamine (Gal NAc)-α-2,6-sialyltransferase I (ST6GalNAcI) polypeptide. The method comprises the steps of producing the sialyltransferase polypeptide in a Chinese Hamster Ovary (CHO) cell and purifying the polypeptide with a combination of chromatography steps. The method results in high yield of sialyltransferase polypeptide which is highly pure and active. The obtained sialyltransferase, especially ST6GalNAcI, can be employed for the glycosylation of therapeutic proteins such as G-CSF.
Claims20
A method of producing a sialyltransferase polypeptide, which comprises the steps of:
- a) expressing a sialyltransferase polypeptide in Chinese hamster ovary cells;
- collecting the cell culture medium containing the expressed sialyltransferase polypeptide, wherein an expression cassette encoding an erythropoietin (EPO) signal sequence and a sialyltransferase polypeptide sequence is used for expressing the sialyltransferase polypeptide in Chinese hamster ovary cells;
- b) purifying the sialyltransferase polypeptide from the cell culture medium by subjecting the cell culture medium to (i) two affinity chromatography steps or one affinity chromatography step and one mixed-mode chromatography step, (ii) one anion exchange chromatography step and (iii) one cation exchange chromatography step;
- and
- c) obtaining an eluate comprising the purified sialyltransferase polypeptide.
Dependencies are inferred from the claim text.