Methods to obtain recombinant proteins with increased sialylation from cells that express adenovirus e1a protein, and proteins obtained thereby

Abstract

Provided are compositions comprising one or more isoforms of an erythropoietin ('EPO') comprising glycans linked thereto, wherein the glycans have Lewis x structures and on average at least six sialic acid moieties per EPO molecule. Further provided are methods for obtaining a composition comprising one or more isoforms of EPO comprising glycans linked thereto, wherein the glycans comprise on average at least six sialic acids per EPO molecule and from zero to two Lewis x structures, the method comprising: a) providing a eukaryotic cell containing a nucleic acid sequence encoding an adenoviral E1A protein in expressible format and a nucleic acid encoding EPO in expressible format, wherein the cell further contains a nucleic acid sequence encoding a sialyltransferase, e.g., an α-2,6-sialyltransferase or an α-2,3-sialyltransferase, under control of a heterologous promoter; b) culturing the cell in a serum-free culture medium and allowing expression of EPO in the cell; c) harvesting the expressed EPO from the cell and/or from the culture medium; and d) purifying and fractionating the EPO to obtain fractions that have an increased average sialic acid content of the N-linked glycans per EPO molecule, to obtain a composition comprising one or more isoforms of an EPO comprising glycans linked thereto, wherein the glycans comprise on average at least six sialic acids per EPO molecule and from zero to two Lewis x structures.