Overexpression of N-glycosylation pathway regulators to modulate glycosylation of recombinant proteins

A method of decreasing the high mannose glycoform content of a recombinant protein during a mammalian cell culture process, compared to a culture in which mammalian cells are not subject to a transfection to overexpress proteins involved in N-glycosylation, the method comprising:

• transfecting a mammalian host cell to overexpress a protein that is involved in an N-glycosylation pathway, wherein the protein involved in the N-glycosylation pathway is selected from the group consisting of N-acetyl-glucosaminyltransferase-1 (encoded by Mgat1);
• N-acetyl-glucosaminyltransferase-2 (encoded by Mgat2);
• a UDP-Galactose transporter encoded by Slc35a2;
• and combinations thereof;
• growing the transfected mammalian host cell, thereby producing transfected mammalian host cells;
• and
• either
• (a) subjecting the transfected mammalian host cells to a temperature shift;
• or
• (b) inducing the transfected mammalian host cells into cell growth arrest by L-asparagine starvation followed by perfusion with a serum-free perfusion media having an L-asparagine concentration of 5 mM or less,
• whereby said high mannose glycoform content of the recombinant protein is decreased.