Process for manipulating the level of glycan content of a glycoprotein

Abstract

The present invention provides a method for manipulating the fucosylated glycan content on a recombinant protein.

What is claimed is: 
     
         1 .- 25  (canceled) 
     
     
         26 . A recombinant protein produced by a method for manipulating the fucosylated glycan content on a recombinant protein comprising
 inoculating a bioreactor with mammalian host cells expressing the recombinant protein,   culturing the mammalian host cells in a serum free, chemically defined cell culture medium;   wherein the cell culture medium includes from 10 to 100 ppb copper and from 50 to 1000 nM manganese. at pH 7.0,   harvesting the recombinant protein produced by the host cell,   
       wherein the level of afucosylated glycans on the recombinant protein increases compared to the afucosylated glycan level obtained in the same cell culture medium at a lower pH. 
     
     
         27 . A recombinant protein according to  claim 26  that is purified. 
     
     
         28 . The recombinant protein according to  claim 26 , formulated into a pharmaceutically acceptable formulation. 
     
     
         29 . The recombinant protein of  claim 26 , wherein there is also an increase in the level of β-galactosylation on the recombinant protein. 
     
     
         30 . The recombinant protein of  claim 26 , wherein the concentration of copper is 100 ppb. 
     
     
         31 . The recombinant protein of  claim 26 , wherein the concentration of manganese is 1000 nM. 
     
     
         32 . The recombinant protein of  claim 26 , wherein the fucosylated glycan content is manipulated to influence the level old afuscosylated and β-galactosylated glycans without impacting cell culture performance. 
     
     
         33 . The recombinant protein of  claim 26 , wherein the method further comprises a temperature shift. 
     
     
         34 . The recombinant protein of  claim 33 , wherein the temperature shift is from 36° C. to 31° C. 
     
     
         35 . The recombinant protein of  claim 33 , wherein the temperature shift occurs at the transition between the growth phase and production phase. 
     
     
         36 . The recombinant protein of  claim 33 , wherein the temperature shift occurs during the production phase. 
     
     
         37 . The recombinant protein of  claim 26 , wherein the mammalian host cell expressing the recombinant protein is cultured in a batch culture, fed-batch culture, perfusion culture, or combinations thereof. 
     
     
         38 . The recombinant protein of  claim 37 , wherein the culture is a perfusion culture. 
     
     
         39 . The recombinant protein of  claim 38 , wherein perfusion comprises continuous perfusion. 
     
     
         40 . The recombinant protein of  claim 38 , wherein the rate of perfusion is constant. 
     
     
         41 . The recombinant protein of  claim 38 , wherein the perfusion is performed at a rate of less than or equal to 1.0 working volumes per day. 
     
     
         42 . The recombinant protein of  claim 38 , wherein the perfusion is accomplished by alternating tangential flow. 
     
     
         43 . The recombinant protein of  claim 26 , wherein the bioreactor has a capacity of at least 500 L. 
     
     
         44 . The recombinant protein of  claim 26 , wherein the bioreactor has a capacity of at least 500 L to 2000 L. 
     
     
         45 . The recombinant protein of  claim 26 , wherein the bioreactor has a capacity of at least 1000 L to 2000 L. 
     
     
         46 . The recombinant protein of  claim 26 , wherein the bioreactor is inoculated with at least 0.5×10 6  cells/mL. 
     
     
         47 . The recombinant protein of  claim 26 , wherein the serum-free chemically defined cell culture medium is a perfusion cell culture medium. 
     
     
         48 . The recombinant protein of  claim 26 , wherein the mammalian host cells are Chinese Hamster Ovary (CHO) cells. 
     
     
         49 . The recombinant protein of  claim 26 , wherein the recombinant protein is a glycoprotein.