Methods to control protein heterogeneity

Abstract

The instant invention relates to the field of protein production, and in particular to compositions and processes for controlling and limiting the heterogeneity of proteins expressed in host cells.

We claim: 
     
         1 . A process for producing a recombinantly-expressed immunoglobulin, comprising culturing a mammalian cell which recombinantly expresses the immunoglobulin during a production stage in a cell culture media comprising asparagine, thereby producing the recombinantly-expressed immunoglobulin,
 wherein the level of agalactosyl fucosylated biantennary oligosaccharides (sum of NGA2F and NGA2F−GlcNac) present on the produced immunoglobulin is increased as compared to the level of agalactosyl fucosylated biantennary oligosaccharides (sum of NGA2F and NGA2F−GlcNac) of immunoglobulin produced in cell culture media which does not comprise said asparagine during the production stage; and/or wherein the level of galactose containing fucosylated biantennary oligossacharides (sum of NA1F and NA2F) present on the produced immunoglobulin is decreased as compared to the level of galactose containing fucosylated biantennary oligossacharides (sum of NA1F and NA2F) of immunoglobulin produced in cell culture media which does not comprise said asparagine during the production stage.   
     
     
         2 . The process of  claim 1 , wherein the immunoglobulin is an anti-TNFα antibody. 
     
     
         3 . The process of  claim 1 , wherein the cell which expresses the immunoglobulin is a CHO cell. 
     
     
         4 . The process of  claim 3 , wherein the immunoglobulin is adalimumab. 
     
     
         5 . The process of  claim 4 , wherein the cell culture media comprises asparagine at a concentration of between 0.4 g/L to 2.0 g/L during the production stage. 
     
     
         6 . The process of  claim 4 , wherein the cell culture media comprises asparagine at a concentration of at least 0.4 g/L, at least 0.8 g/L, at least 1.2 g/L, at least 1.6 g/L or at least 2.0 g/L. 
     
     
         7 . The process of  claim 4 , wherein the cell culture media further comprises glutamine. 
     
     
         8 . The process of  claim 7 , wherein the cell culture media comprises glutamine at a concentration of at least 0.4 g/L. 
     
     
         9 . The process of  claim 4 , wherein the level of agalactosyl fucosylated biantennary oligosaccharides (sum of NGA2F and NGA2F−GlcNAc) present on the produced immunoglobulin is 64%-88%, 70%-88% or 75%-85%; and/or wherein the level of fucosylated biantennary oligosaccharides (sum of NA1F and NA2F) present on the produced immunoglobulin is 1%-30%, 2%-25%, 5%-20%, 5%-15%, 10%-20% or 27%-31%. 
     
     
         10 . The process of  claim 4 , wherein the process is a fed batch process. 
     
     
         11 . The process of  claim 4 , wherein the cell culture media further comprises a yeast hydrolysate and/or a plant hydrolysate; optionally, wherein the yeast hydrolysate is selected from the group consisting of Bacto TC Yeastolate, HyPep Yeast Extract and UF Yeast Hydrolysate; and/or wherein the plant hydrolysate is selected from the group consisting of a soy hydrolysate, a wheat hydrolysate, a rice hydrolysate, a cotton seed hydrolysate, a pea hydrolysate, a corn hydrolysate, a potato hydrolysate, BBL Phytone Peptone, HyPep 1510, SE50 MAF-UF, UF Soy Hydrolysate, Wheat Peptone E1, HyPep 4601 and Proyield WGE80M Wheat. 
     
     
         12 . The process of  claim 11 , wherein the yeast hydrolysate is present in the cell culture media at a concentration of between 2 g/L to 11 g/L and/or wherein the plant hydrolysate is present in the cell culture media at a concentration of between 2 g/L to 15 g/L. 
     
     
         13 . The process of  claim 4 , wherein the cell culture media is a chemically defined cell culture media. 
     
     
         14 . The process of  claim 4 , wherein the production stage initiates at an initial viable cell density of approximately 0.5×10 6  cells/mL. 
     
     
         15 . The process of  claim 1 , further comprising collecting and isolating the recombinantly-expressed immunoglobulin. 
     
     
         16 . The process of  claim 4 , wherein the asparagine is present at a concentration of less than or equal to 26 mM. 
     
     
         17 . A process for producing a recombinantly-expressed immunoglobulin, comprising culturing a mammalian cell which recombinantly expresses the immunoglobulin in a cell culture media comprising at least 0.8 g/L of asparagine, thereby producing the recombinantly-expressed immunoglobulin,
 wherein the level of agalactosyl fucosylated biantennary oligosaccharides (sum of NGA2F and NGA2F−GlcNac) present on the produced immunoglobulin is increased as compared to the level of agalactosyl fucosylated biantennary oligosaccharides (sum of NGA2F and NGA2F−GlcNac) of immunoglobulin produced in cell culture media which does not comprise said asparagine; and/or wherein the level of galactose containing fucosylated biantennary oligossacharides (sum of NA1F and NA2F) present on the produced immunoglobulin is decreased as compared to the level of galactose containing fucosylated biantennary oligossacharides (sum of NA1F and NA2F) of immunoglobulin produced in cell culture media which does not comprise said asparagine.   
     
     
         18 . The process of  claim 17 , wherein the immunoglobulin is an anti-TNFα antibody. 
     
     
         19 . The process of  claim 17 , wherein the cell which expresses the immunoglobulin is a CHO cell. 
     
     
         20 . The process of  claim 19 , wherein the immunoglobulin is adalimumab. 
     
     
         21 . The process of  claim 20 , wherein the cell culture media comprises asparagine at a concentration of at least 1.2 g/L, at least 1.6 g/L or at least 2.0 g/L. 
     
     
         22 . The process of  claim 20 , wherein the cell culture media comprises asparagine at a concentration of between 0.8 g/L to 2.0 g/L. 
     
     
         23 . The process of  claim 20 , wherein the cell culture media further comprises glutamine. 
     
     
         24 . The process of  claim 23 , wherein the cell culture media comprises glutamine at a concentration of at least 0.4 g/L. 
     
     
         25 . The process of  claim 20 , wherein the level of agalactosyl fucosylated biantennary oligosaccharides (sum of NGA2F and NGA2F−GlcNAc) present on the produced immunoglobulin is 64%-88%, 70%-88% or 75%-85%; and/or wherein the level of fucosylated biantennary oligosaccharides (sum of NA1F and NA2F) present on the produced immunoglobulin is 1%-30%, 2%-25%, 5%-20%, 5%-15%, 10%-20% or 27%-31%. 
     
     
         26 . The process of  claim 20 , wherein the process is a fed batch process. 
     
     
         27 . The process of  claim 20 , wherein the culture media further comprises a yeast hydrolysate and/or a plant hydrolysate; optionally, wherein the yeast hydrolysate is selected from the group consisting of Bacto TC Yeastolate, HyPep Yeast Extract and UF Yeast Hydrolysate; and/or wherein the plant hydrolysate is selected from the group consisting of a soy hydrolysate, a wheat hydrolysate, a rice hydrolysate, a cotton seed hydrolysate, a pea hydrolysate, a corn hydrolysate, a potato hydrolysate, BBL Phytone Peptone, HyPep 1510, SE50 MAF-UF, UF Soy Hydrolysate, Wheat Peptone E1, HyPep 4601 and Proyield WGE80M Wheat. 
     
     
         28 . The process of  claim 27 , wherein the yeast hydrolysate is present in the cell culture media at a concentration of between 2 g/L to 11 g/L and/or wherein the plant hydrolysate is present in the cell culture media at a concentration of between 2 g/L to 15 g/L. 
     
     
         29 . The process of  claim 20 , wherein the cell culture media is a chemically defined cell culture media. 
     
     
         30 . The process of  claim 17 , further comprising collecting and isolating the recombinantly-expressed immunoglobulin.