Control of Protein Glycosylation by Culture Medium Supplementation and Cell Culture Process Parameters

Abstract

The present invention pertains to a cell culture medium comprising media supplements that are shown to control recombinant protein glycosylation and/or cell culture in a controlled or modulated (shifted) temperature to control recombinant protein glycosylation and/or cell culture with controlled or modulated seed density to control recombinant protein glycosylation, and methods of using thereof. The present invention further pertains to a method of controlling or manipulating glycosylation of a recombinant protein of interest in a large scale cell culture.

1 . A method of altering the glycosylation pattern of a recombinant glycoprotein produced in cell culture comprising:
 culturing eukaryotic cells engineered to express a recombinant glycoprotein of interest in a cell culture medium, wherein the cell culture medium is supplemented with an additive comprising one or more of mycophenolic acid, mycophenolic acid acyl glucuronide, insulin, copper (II) sulfate, hypoxanthine, thymidine, guanine, glucosamine, or galactose;   wherein the glycosylation pattern of the recombinant glycoprotein of interest is altered relative to the same recombinant glycoprotein produced by the same cells in the same cell culture medium without the additive.   
     
     
         2 . A method of altering the glycosylation pattern of a recombinant glycoprotein produced in cell culture comprising:
 culturing eukaryotic cells engineered to express a recombinant glycoprotein of interest in a cell culture medium, wherein the cell culture medium is supplemented with an additive comprising one or more of mycophenolic acid, mycophenolic acid acyl glucuronide, insulin, copper (II) sulfate, hypoxanthine, thymidine, guanine, glucosamine, or galactose;   wherein the glycosylation pattern of the recombinant glycoprotein of interest is altered to better resemble the glycosylation pattern of a reference sample of the glycoprotein.   
     
     
         3 . A method of altering the glycosylation pattern of a recombinant glycoprotein produced in cell culture comprising:
 supplementing the culture medium of a cell culture of eukaryotic cells engineered to express a recombinant glycoprotein of interest with an additive comprising one or more of mycophenolic acid, mycophenolic acid acyl glucuronide, insulin, copper (II) sulfate, hypoxanthine, thymidine, guanine, glucosamine, or galactose;   wherein the glycosylation pattern of the recombinant glycoprotein of interest is altered relative to the same recombinant glycoprotein produced by the same cells in the same cell culture medium without the additive.   
     
     
         4 . A method of altering the glycosylation pattern of a recombinant glycoprotein produced in cell culture comprising:
 supplementing the culture medium of a cell culture of eukaryotic cells engineered to express a recombinant glycoprotein of interest with an additive comprising one or more of mycophenolic acid, mycophenolic acid acyl glucuronide, insulin, copper (II) sulfate, hypoxanthine, thymidine, guanine, glucosamine, or galactose;   wherein the glycosylation pattern of the recombinant glycoprotein of interest is altered to better resemble the glycosylation pattern of a reference sample of the glycoprotein.   
     
     
         5 . The method of  claim 1 , further comprising recovering the recombinant glycoprotein of interest from the cell culture. 
     
     
         6 . The method of  claim 1 , wherein the alteration of the glycosylation pattern of the recombinant glycoprotein of interest comprises one or more of a reduced level of afucosylation, a reduced level of galactosylation, a reduced level of galactose-alpha-1,3-galactose (α-gal), a reduced level of N-glycolylneuraminic acid (NGNA), reduced FcγRIIIa binding, reduced antibody-dependent cell-mediated cytotoxicity, an increased N-glycan charge, or increased level of afucosylation. 
     
     
         7 . The method of  claim 1 , wherein the alteration of the glycosylation pattern of the recombinant glycoprotein of interest is achieved while minimizing one or more undesirable side effects. 
     
     
         8 . The method of  claim 7 , wherein the alteration of the glycosylation pattern is achieved without substantially increasing the level of α-gal. 
     
     
         9 . The method of  claim 7 , wherein the alteration of the glycosylation pattern is achieved without substantially reducing sialic acid levels. 
     
     
         10 . The method of  claim 7 , wherein the alteration of the glycosylation pattern is achieved while maintaining an acceptable culture density, an acceptable cell viability level, or both an acceptable culture density and an acceptable cell viability level. 
     
     
         11 . The method of  claim 1 , wherein the cell culture comprises a growth phase and a protein production phase, and wherein the additive is introduced into the culture medium before or at the same time as the initiation of the protein production phase. 
     
     
         12 . The method of  claim 1 , wherein the cell culture is conducted in a shake flask. 
     
     
         13 . The method of  claim 1 , wherein the cell culture is conducted in a stirred-tank bioreactor. 
     
     
         14 . The method of  claim 13 , wherein the bioreactor has a volume of between about 500 liters and about 30,000 liters. 
     
     
         15 - 172 . (canceled) 
     
     
         173 . The method of  claim 1 , wherein the additive comprises mycophenolic acid acyl glucuronide. 
     
     
         174 . The method of  claim 173 , wherein the supplemented cell culture medium comprises between about 1 μM and about 50 μM mycophenolic acid acyl glucuronide. 
     
     
         175 . The method of  claim 174 , wherein the supplemented cell culture medium comprises between about 1 μM and about 30 μM mycophenolic acid acyl glucuronide. 
     
     
         176 . The method of  claim 173 , wherein the mycophenolic acid acyl glucuronide is introduced into the cell culture medium as part of a feed medium. 
     
     
         177 . The method of  claim 173 , wherein the mycophenolic acid acyl glucuronide is introduced into the cell culture medium as one or more boli from a distinct stock solution. 
     
     
         178 . The method of  claim 173 , wherein the method further comprises controlling or modulating cell culture temperature. 
     
     
         179 . The method of  claim 178 , wherein the supplemented cell culture medium comprises between about 1 μM and about 50 μM of mycophenolic acid acyl glucuronide. 
     
     
         180 . The method of  claim 179 , wherein the supplemented cell culture medium comprises about between 1 μM and about 30 μM of mycophenolic acid acyl glucuronide. 
     
     
         181 . The method of  claim 178 , wherein the mycophenolic acid acyl glucuronide is introduced into the cell culture medium as part of a feed medium. 
     
     
         182 . The method of  claim 178 , wherein the mycophenolic acid acyl glucuronide is introduced at the same time as the cell culture temperature is controlled or modulated. 
     
     
         183 . The method of  claim 178 , wherein the mycophenolic acid acyl glucuronide is introduced at a different time than the time when the cell culture temperature is controlled or modulated. 
     
     
         184 . The method of  claim 178 , wherein the method further comprises controlling or modulating cell culture seed density. 
     
     
         185 . The method of  claim 184 , wherein the supplemented cell culture medium comprises between about 1 μM and about 50 μM of mycophenolic acid acyl glucuronide. 
     
     
         186 . The method of  claim 185 , wherein the supplemented cell culture medium comprises about between 1 μM and about 30 μM of mycophenolic acid. 
     
     
         187 . The method of  claim 184 , wherein the mycophenolic acid acyl glucuronide is introduced into the cell culture medium as part of a feed medium. 
     
     
         188 . The method of  claim 184 , wherein the mycophenolic acid acyl glucuronide is introduced into the cell culture medium as one or more boli from a distinct stock solution. 
     
     
         189 . The method of  claim 184 , wherein the mycophenolic acid acyl glucuronide is introduced at the same time as the cell culture temperature is controlled or modulated. 
     
     
         190 . The method of  claim 184 , wherein the mycophenolic acid acyl glucuronide is introduced at a different time than the time when the cell culture temperature is controlled or modulated. 
     
     
         191 . The method of  claim 178 , wherein the mycophenolic acid acyl glucuronide is introduced into the cell culture medium as one or more boli from a distinct stock solution.