Methods of cell culture

Abstract

Polypeptide preparations having target levels of glycans, and methods of producing such polypeptide preparations using ammonium and/or lysine, are described.

1 . A method of producing a recombinant protein preparation having a target value of one or more of high mannose glycans, galactosylated glycans, and fucosylated glycans, the method comprising:
 (a) providing a cell genetically engineered to express a recombinant protein;   (b) culturing the cell in a culture medium comprising ammonium under conditions in which the cell expresses the recombinant protein; and   (c) harvesting a preparation of the recombinant protein produced by the cell, wherein the preparation has the target value of one or more of high mannose glycans, galactosylated glycans, and fucosylated glycans.   
     
     
         2 . The method of  claim 1 , wherein the recombinant protein is a recombinant therapeutic protein. 
     
     
         3 . The method of  claim 1 , wherein the recombinant protein is a recombinant therapeutic antibody. 
     
     
         4 . The method of  claim 1 , wherein the target value is a level of one or more of high mannose glycans, galactosylated glycans, and fucosylated glycans in a reference therapeutic product. 
     
     
         5 . The method of  claim 1 , wherein the target value is a level of one or more of high mannose glycans, galactosylated glycans, and fucosylated glycans in a reference therapeutic antibody product. 
     
     
         6 . The method of  claim 1 , wherein the target value is a predetermined pharmaceutical product specification. 
     
     
         7 . The method of  claim 4 , wherein the reference therapeutic product is selected from the group consisting of: abatacept, abciximab, adalimumab, aflibercept, alefacept, alemtuzumab, basiliximab, bevacizumab, belatacept, certolizumab, cetuximab, daclizumab, eculizumab, efalizumab, entanercept, gemtuzumab, ibritumomab, infliximab, muromonab-CD3, natalizumab, omalizumab, palivizumab; panitumumab, ranibizumab, rilonacept, rituximab, tositumomab, and trastuzumab. 
     
     
         8 . The method of  claim 1 , further comprising evaluating a level of one or more of high mannose glycans, galactosylated glycans, and fucosylated glycans in the recombinant protein preparation. 
     
     
         9 . The method of  claim 1 , wherein the target value is one or more of:
 (a) 0.1% to 20% high mannose glycans,   (b) 1% to 95% galactosylated glycans, and   (c) 70% to 100% fucosylated glycans.   
     
     
         10 . The method of  claim 1 , wherein the culture medium comprises an elevated level of ammonium. 
     
     
         11 . The method of  claim 10 , wherein the elevated level of ammonium is 1 mM to 50 mM ammonium. 
     
     
         12 . The method of  claim 10 , wherein the target value of the high mannose glycans is higher than a corresponding level in a preparation produced by culturing the cell in the medium not comprising the elevated level of ammonium. 
     
     
         13 . The method of  claim 12 , wherein the target value is higher than the corresponding level by at least 10% of the corresponding level. 
     
     
         14 . The method of  claim 10 , wherein the target value of the one or more of galactosylated glycans and fucosylated glycans is lower than a corresponding level in a preparation produced by culturing the cell in the medium not comprising the elevated level of ammonium. 
     
     
         15 . The method of  claim 14 , wherein the target value is lower than the corresponding level by at least 10% of the corresponding level. 
     
     
         16 . The method of  claim 1 , wherein the culture medium further comprises one or more of glucosamine, lysine, copper, putrescine, glucose, a growth factor, a vitamin, a lipid, a peptone, and DMSO. 
     
     
         17 . A method of producing a recombinant protein preparation, the method comprising:
 (a) providing a target value of one or more of high mannose glycans, galactosylated glycans, and fucosylated glycans;   (b) providing a cell genetically engineered to express a recombinant protein;   (c) culturing the cell in a culture medium comprising ammonium under conditions in which the cell expresses the recombinant protein;   (d) harvesting a preparation of the recombinant protein produced by the cell; and   (e) formulating the preparation into a drug product if the preparation meets the target value of the one or more of high mannose glycans, galactosylated glycans, and fucosylated glycans.   
     
     
         18 . The method of  claim 17 , further comprising evaluating a level of one or more of high mannose glycans, galactosylated glycans, and fucosylated glycans in the recombinant protein preparation. 
     
     
         19 . The method of  claim 17 , wherein the culture medium further comprises one or more of glucosamine, lysine, copper, putrescine, glucose, a growth factor, a vitamin, a lipid, a peptone, and DMSO. 
     
     
         20 . A method of increasing a level of one or more of high mannose glycans, afucosylated glycans, and G0F glycans in a recombinant protein preparation, the method comprising:
 (a) providing a cell genetically engineered to express a recombinant protein;   (b) culturing the cell in a culture medium comprising an elevated level of ammonium under conditions in which the cell expresses the recombinant protein; and   (c) harvesting a preparation of the recombinant protein produced by the cell, wherein the preparation has an increased level of one or more of high mannose glycans, afucosylated glycans, and G0F glycans relative to a corresponding level in a preparation of the recombinant protein produced using the medium not comprising the elevated level of ammonium.   
     
     
         21 . The method of  claim 20 , further comprising measuring a level of one or more of high mannose glycans, afucosylated glycans, and G0F glycans. 
     
     
         22 . The method of  claim 20 , wherein the increased level of the one or more of high mannose glycans, afucosylated glycans, and G0F glycans is higher than the corresponding level by at least 10% of the corresponding level. 
     
     
         23 . The method of  claim 20 , wherein the culture medium further comprises one or more of glucosamine, lysine, copper, putrescine, glucose, a growth factor, a vitamin, a lipid, a peptone, and DMSO. 
     
     
         24 . A method of decreasing a level of one or more of galactosylated glycans and fucosylated glycans in a recombinant protein preparation, the method comprising:
 (a) providing a cell genetically engineered to express a recombinant protein;   (b) culturing the cell in a culture medium comprising an elevated level of ammonium under conditions in which the cell expresses the recombinant protein; and   (c) harvesting a preparation of the recombinant protein produced by the cell, wherein the preparation has a decreased level of one or more of galactosylated glycans and fucosylated glycans relative to corresponding level in a preparation of the recombinant protein produced using the medium not comprising the elevated level of ammonium.   
     
     
         25 . The method of  claim 24 , wherein the fucosylated glycans comprise one or more of G1F glycans and G2F glycans. 
     
     
         26 . The method of  claim 24 , further comprising measuring a level of one or more of galactosylated glycans and fucosylated glycans. 
     
     
         27 . The method of  claim 26 , wherein the decreased level of the one or more of galactosylated glycans and fucosylated glycans is lower than the corresponding level by at least 10% of the corresponding level. 
     
     
         28 . The method of  claim 26 , wherein the culture medium further comprises one or more of glucosamine, lysine, copper, putrescine, glucose, a growth factor, a vitamin, a lipid, a peptone, and DMSO. 
     
     
         29 . The method of any one of  claims 1 ,  17 ,  20 , and  24 , wherein the culturing step comprises a first stage and a second stage. 
     
     
         30 . The method of  claim 29 , wherein the first stage comprises culturing the cell in the culture medium comprising a first level of ammonium, and the second stage comprises culturing the cell in the culture medium comprising a second level of ammonium. 
     
     
         31 . The method of  claim 30 , wherein the second level is 1 mM to 50 mM ammonium. 
     
     
         32 . The method of  claim 30 , wherein the first stage comprises culturing the cell in the first level of ammonium for 1 to 8 days. 
     
     
         33 . The method of  claim 32 , wherein the second stage comprises culturing the cell in the second level of ammonium for 1 to 12 days. 
     
     
         34 . The method of any one of  claims 1 ,  17 ,  20 , and  24 , wherein the cell is a CHO cell. 
     
     
         35 . A method of producing a recombinant protein preparation having a target value of one or more of high mannose glycans, galactosylated glycans, fucosylated glycans, and sialylated glycans, the method comprising:
 (a) providing a cell genetically engineered to express a recombinant protein;   (b) culturing the cell in a culture medium comprising lysine under conditions in which the cell expresses the recombinant protein; and   (c) harvesting a preparation of the recombinant protein produced by the cell, wherein the preparation has the target value of one or more of high mannose glycans, galactosylated glycans, fucosylated glycans, and sialylated glycans.   
     
     
         36 . The method of  claim 35 , wherein the recombinant protein is a recombinant therapeutic protein. 
     
     
         37 . The method of  claim 35 , wherein the recombinant protein is a recombinant therapeutic antibody. 
     
     
         38 . The method of  claim 35 , wherein the target value is a level of one or more of high mannose glycans, galactosylated glycans, fucosylated glycans, and sialylated glycans in a reference therapeutic product. 
     
     
         39 . The method of  claim 35 , wherein the target value is a level of one or more of high mannose glycans, galactosylated glycans, fucosylated glycans, and sialylated glycans in a reference therapeutic antibody product. 
     
     
         40 . The method of  claim 35 , wherein the target value is a predetermined pharmaceutical product specification. 
     
     
         41 . The method of  claim 38 , wherein the reference therapeutic product is selected from the group consisting of: abatacept, abciximab, adalimumab, aflibercept, alefacept, alemtuzumab, basiliximab, bevacizumab, belatacept, certolizumab, cetuximab, daclizumab, eculizumab, efalizumab, entanercept, gemtuzumab, ibritumomab, infliximab, muromonab-CD3, natalizumab, omalizumab, palivizumab; panitumumab, ranibizumab, rilonacept, rituximab, tositumomab, and trastuzumab. 
     
     
         42 . The method of  claim 35 , further comprising evaluating a level of one or more of high mannose glycans, galactosylated glycans, fucosylated glycans, and sialylated glycans in the recombinant protein preparation. 
     
     
         43 . The method of  claim 35 , wherein the target value is one or more of:
 (a) 0.1% to 20% high mannose glycans,   (b) 1% to 95% galactosylated glycans,   (c) 70% to 100% fucosylated glycans, and   (d) 0.1% to 90% sialylated glycans.   
     
     
         44 . The method of  claim 35 , wherein the culture medium comprises an elevated level of lysine. 
     
     
         45 . The method of  claim 44 , wherein the elevated level of lysine is 2 g/L to 30 g/L lysine. 
     
     
         46 . The method of  claim 44 , wherein the target value of the high mannose glycans is higher than a corresponding level in a preparation produced by culturing the cell in the medium not comprising the elevated level of lysine. 
     
     
         47 . The method of  claim 46 , wherein the target value is higher than the corresponding level by at least 10% of the corresponding level. 
     
     
         48 . The method of  claim 44 , wherein the target value of the one or more of galactosylated glycans, sialylated glycans, and fucosylated glycans is lower than a corresponding level in a preparation produced by culturing the cell in the medium not comprising the elevated level of lysine. 
     
     
         49 . The method of  claim 48 , wherein the target value is lower than the corresponding level by at least 10% of the corresponding level. 
     
     
         50 . The method of  claim 35 , wherein the culture medium further comprises one or more of glucosamine, ammonium, copper, putrescine, glucose, a growth factor, a vitamin, a lipid, a peptone, and DMSO. 
     
     
         51 . A method of producing a recombinant protein preparation, the method comprising:
 (a) providing a target value of one or more of high mannose glycans, galactosylated glycans, fucosylated glycans, and sialylated glycans;   (b) providing a cell genetically engineered to express a recombinant protein;   (c) culturing the cell in a culture medium comprising lysine under conditions in which the cell expresses the recombinant protein;   (d) harvesting a preparation of the recombinant protein produced by the cell; and   (e) formulating the preparation into a drug product if the preparation meets the target value of the one or more of high mannose glycans, galactosylated glycans, fucosylated glycans, and sialylated glycans.   
     
     
         52 . The method of  claim 51 , further comprising evaluating a level of one or more of high mannose glycans, galactosylated glycans, fucosylated glycans, and sialylated glycans in the recombinant protein preparation. 
     
     
         53 . The method of  claim 51 , wherein the culture medium further comprises one or more of glucosamine, ammonium, copper, putrescine, glucose, a growth factor, a vitamin, a lipid, a peptone, and DMSO. 
     
     
         54 . A method of increasing a level of one or more of high mannose glycans, afucosylated glycans, and G0F glycans in a recombinant protein preparation, the method comprising:
 (a) providing a cell genetically engineered to express a recombinant protein;   (b) culturing the cell in a culture medium comprising an elevated level of lysine under conditions in which the cell expresses the recombinant protein; and   (c) harvesting a preparation of the recombinant protein produced by the cell, wherein the preparation has an increased level of one or more of high mannose glycans, afucosylated glycans, and G0F glycans relative to a corresponding level in a preparation of the recombinant protein produced using the medium not comprising the elevated level of lysine.   
     
     
         55 . The method of  claim 54 , further comprising measuring a level of one or more of high mannose glycans, afucosylated glycans, and G0F glycans. 
     
     
         56 . The method of  claim 55 , wherein the increased level of the one or more of high mannose glycans, afucosylated glycans, and G0F glycans is higher than the corresponding level by at least 10% of the corresponding level. 
     
     
         57 . The method of  claim 54 , wherein the culture medium further comprises one or more of glucosamine, ammonium, copper, putrescine, glucose, a growth factor, a vitamin, a lipid, a peptone, and DMSO. 
     
     
         58 . A method of decreasing a level of one or more of galactosylated glycans, fucosylated glycans, and sialylated glycans in a recombinant protein preparation, the method comprising:
 (a) providing a cell genetically engineered to express a recombinant protein;   (b) culturing the cell in a culture medium comprising an elevated level of lysine under conditions in which the cell expresses the recombinant protein; and   (c) harvesting a preparation of the recombinant protein produced by the cell, wherein the preparation has a decreased level of one or more of galactosylated glycans, fucosylated glycans, and sialylated glycans relative to a corresponding level in a preparation of the recombinant protein produced using the medium not comprising the elevated level of lysine.   
     
     
         59 . The method of  claim 58 , wherein the fucosylated glycans comprise one or more of G1F glycans and G2F glycans. 
     
     
         60 . The method of  claim 58 , further comprising measuring a level of one or more of galactosylated glycans, fucosylated glycans, and sialylated glycans. 
     
     
         61 . The method of  claim 58 , wherein the decreased level of one or more of galactosylated glycans, fucosylated glycans, and sialylated glycans is lower than the corresponding level by at least 10% of the corresponding level. 
     
     
         62 . The method of  claim 58 , wherein the culture medium further comprises one or more of glucosamine, ammonium, copper, putrescine, glucose, a growth factor, a vitamin, a lipid, a peptone, and DMSO. 
     
     
         63 . The method of any one of  claims 35 ,  51 ,  54 , and  58 , wherein the culturing step comprises a first stage and a second stage. 
     
     
         64 . The method of  claim 63 , wherein the first stage comprises culturing the cell in the culture medium comprising a first level of lysine, and the second stage comprises culturing the cell in the culture medium comprising a second level of lysine. 
     
     
         65 . The method of  claim 64 , wherein the second level is 2 g/L to 30 g/L lysine. 
     
     
         66 . The method of  claim 64 , wherein the first stage comprises culturing the cell in the first level of lysine for 1 to 8 days. 
     
     
         67 . The method of  claim 66 , wherein the second stage comprises culturing the cell in the second level of lysine for 1 to 12 days. 
     
     
         68 . The method of any one of  claims 35 ,  51 ,  54 , and  58 , wherein the cell is a CHO cell. 
     
     
         69 . A method of producing a recombinant protein preparation having a target value of one or more of high mannose glycans, galactosylated glycans, and fucosylated glycans, the method comprising:
 (a) providing a cell genetically engineered to express a recombinant protein;   (b) culturing the cell in a culture medium comprising a pH modifying agent under conditions in which the cell expresses the recombinant protein; and   (c) harvesting a preparation of the recombinant protein produced by the cell, wherein the preparation has the target value of one or more of high mannose glycans, galactosylated glycans, and fucosylated glycans.