Methods to control protein heterogeneity

Abstract

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

1 . A method for controlling the oligosaccharide distribution of a recombinantly-expressed immunoglobulin comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 2 and a light chain variable region comprising the sequence of SEQ ID NO: 7, comprising
 supplementing during a production stage a cell culture media used in the recombinant expression of said immunoglobulin with an asparagine supplement to achieve an asparagine concentration in the media of at least 0.4 g/L, and   assessing the oligosaccharide distribution of the recombinantly-expressed immunoglobulin, thereby controlling the oligosaccharide distribution of the recombinantly-expressed immunoglobulin,   wherein the level of agalactosyl fucosylated biantennary oligosaccharides (sum of NGA2F and NGA2F-GlcNac) present on the recombinantly-expressed immunoglobulin is increased as compared to the level of agalactosyl fucosylated biantennary oligosaccharides (sum of NGA2F and NGA2F-GlcNac) of immunoglobulin recombinantly expressed in cell culture media which is not supplemented with said asparagine supplement during the production stage; and/or   wherein the level of galactose containing fucosylated biantennary oligosaccharides (sum of NA1F and NA2F) present on the recombinantly-expressed immunoglobulin is decreased as compared to the level of galactose containing fucosylated biantennary oligosaccharides (sum of NA1F and NA2F) of immunoglobulin recombinantly expressed in cell culture media which is not supplemented with said asparagine supplement during the production stage.   
     
     
         2 . The method of  claim 1 , wherein the immunoglobulin is adalimumab. 
     
     
         3 . The method of  claim 1 , wherein the recombinantly-expressed immunoglobulin is produced by a CHO cell. 
     
     
         4 . The method of  claim 1 , wherein the cell culture media
 (a) is supplemented with the asparagine supplement to achieve an asparagine concentration of at least 0.6 g/L, 0.8 g/L, 1.0 g/L, 1.2 g/L, 1.4 g/L, 1.6 g/L, 1.8 g/L or 2 g/L, or between 0.4 g/L-1.4 g/L, or between 0.4 g/L to 2 g/L;   (b) further comprises glutamine, optionally, wherein the cell culture media comprises glutamine at a concentration of at least 0.2 g/L or between 0.1 g/L to 4 g/L; and/or   (c) 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; and/or   wherein the yeast hydrolysate is present in the media at a concentration of between 2 g/L to 11 g/L and/or wherein the plant hydrolysate is present in the media at a concentration of between 2 g/L to 15 g/L.   
     
     
         5 - 6 . (canceled) 
     
     
         7 . The method of  claim 1 , wherein assessing the oligosaccharide distribution of the recombinantly-expressed immunoglobulin comprises
 (a) assessing the level of agalactosyl fucosylated biantennary oligosaccharides (sum of NGA2F and NGA2F-GlcNac) present on the recombinantly-expressed immunoglobulin; and/or   (b) assessing the level of galactose containing fucosylated biantennary oligosaccharides (sum of NA1F and NA2F) present on the recombinantly-expressed immunoglobulin;   optionally, wherein the level of agalactosyl fucosylated biantennary oligosaccharides (sum of NGA2F and NGA2F-GlcNAc) present on the recombinantly-expressed 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 recombinantly-expressed immunoglobulin is 1%-30%, 2%-25%, 5%-20%, 5%-15%, 10%-20% or 27%-31%.   
     
     
         8 - 9 . (canceled) 
     
     
         10 . The method of  claim 2 , wherein the method produces at least 2.0 g/L of adalimumab. 
     
     
         11 . The method of  claim 1 , wherein the method is a fed batch process. 
     
     
         12 - 13 . (canceled) 
     
     
         14 . The method of  claim 1 , further comprising collecting and isolating the recombinantly-expressed immunoglobulin. 
     
     
         15 . The method of  claim 1 , wherein the production stage initiates at an initial viable cell density of approximately 0.5×10 6  cells/mL. 
     
     
         16 . A method for controlling the oligosaccharide distribution of a recombinantly-expressed immunoglobulin comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 2 and a light chain variable region comprising the sequence of SEQ ID NO: 7, comprising
 supplementing a cell culture media used in the recombinant expression of said immunoglobulin with an asparagine supplement and a glutamine supplement to achieve a glutamine concentration in the media of at least 1 g/L, and   assessing the oligosaccharide distribution of the recombinantly-expressed immunoglobulin, thereby controlling the oligosaccharide distribution of the recombinantly-expressed immunoglobulin,   wherein the level of agalactosyl fucosylated biantennary oligosaccharides (sum of NGA2F and NGA2F-GlcNac) present on the recombinantly-expressed immunoglobulin is increased as compared to the level of agalactosyl fucosylated biantennary oligosaccharides (sum of NGA2F and NGA2F-GlcNac) of immunoglobulin recombinantly expressed in cell culture media which is not supplemented with said asparagine supplement and said glutamine supplement; and/or   wherein the level of galactose containing fucosylated biantennary oligosaccharides (sum of NA1F and NA2F) present on the recombinantly-expressed immunoglobulin is decreased as compared to the level of galactose containing fucosylated biantennary oligosaccharides (sum of NA1F and NA2F) of immunoglobulin recombinantly expressed in cell culture media which is not supplemented with said asparagine supplement and said glutamine supplement.   
     
     
         17 . The method of  claim 16 , wherein the immunoglobulin is adalimumab. 
     
     
         18 . The method of  claim 16 , wherein the recombinantly-expressed immunoglobulin is produced by a CHO cell. 
     
     
         19 . The method of  claim 16 , wherein the cell culture media
 (a) is supplemented with the asparagine supplement to achieve an asparagine concentration of at least 0.2 g/L, or between 0.1 g/L to 4.0 g/L;   (b) is supplemented with the glutamine supplement to achieve a glutamine concentration of at least 2 g/L; and/or   (c) 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; and/or   wherein the yeast hydrolysate is present in the media at a concentration of between 2 g/L to 11 g/L and/or wherein the plant hydrolysate is present in the media at a concentration of between 2 g/L to 15 g/L.   
     
     
         20 . (canceled) 
     
     
         21 . The method of  claim 16 , wherein assessing the oligosaccharide distribution of the recombinantly-expressed immunoglobulin comprises
 (a) assessing the level of agalactosyl fucosylated biantennary oligosaccharides (sum of NGA2F and NGA2F-GlcNAc) present on the recombinantly-expressed immunoglobulin; and/or   (b) assessing the level of galactose containing fucosylated biantennary oligosaccharides (sum of NA1F and NA2F) present on the recombinantly-expressed immunoglobulin;   optionally, wherein the level of agalactosyl fucosylated biantennary oligosaccharides (sum of NGA2F and NGA2F-GlcNAc) present on the recombinantly-expressed 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 recombinantly-expressed immunoglobulin is 1%-30%, 2%-25%, 5%-20%, 5%-15%, 10%-20% or 27%-31%.   
     
     
         22 - 23 . (canceled) 
     
     
         24 . The method of  claim 16 , wherein the method is a fed batch process. 
     
     
         25 - 26 . (canceled) 
     
     
         27 . A method for controlling the oligosaccharide distribution of a recombinantly-expressed immunoglobulin comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 2 and a light chain variable region comprising the sequence of SEQ ID NO: 7, comprising
 supplementing a cell culture media used in the recombinant expression of said immunoglobulin with an asparagine supplement to achieve an asparagine concentration in the media of at least 0.6 g/L, and   assessing the oligosaccharide distribution of the recombinantly-expressed immunoglobulin, thereby controlling the oligosaccharide distribution of the recombinantly-expressed immunoglobulin,   wherein the level of agalactosyl fucosylated biantennary oligosaccharides (sum of NGA2F and NGA2F-GlcNac) present on the recombinantly-expressed immunoglobulin is increased as compared to the level of agalactosyl fucosylated biantennary oligosaccharides (sum of NGA2F and NGA2F-GlcNac) of immunoglobulin recombinantly expressed in cell culture media which is not supplemented with said asparagine supplement; and/or   wherein the level of galactose containing fucosylated biantennary oligosaccharides (sum of NA1F and NA2F) present on the recombinantly expressed immunoglobulin is decreased as compared to the level of galactose containing fucosylated biantennary oligosaccharides (sum of NA1F and NA2F) of immunoglobulin recombinantly expressed in cell culture media which is not supplemented with said asparagine supplement.   
     
     
         28 . The method of  claim 27 , wherein the immunoglobulin is adalimumab. 
     
     
         29 . The method of  claim 27 , wherein the recombinantly-expressed immunoglobulin is produced by a CHO cell. 
     
     
         30 . The method of  claim 27 , wherein the cell culture media
 (a) is supplemented with the asparagine supplement to achieve an asparagine concentration of at least 0.8 g/L, 1.0 g/L, 1.2 g/L, 1.4 g/L, 1.6 g/L, 1.8 g/L or 2.0 g/L;   (b) further comprises glutamine; optionally, wherein the cell culture media comprises glutamine at a concentration of at least 0.2 g/L or between 0.1 g/L to 4 g/L; and/or   (c) 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; and/or   wherein the yeast hydrolysate is present in the media at a concentration of between 2 g/L to 11 g/L and/or wherein the plant hydrolysate is present in the media at a concentration of between 2 g/L to 15 g/L.   
     
     
         31 - 32 . (canceled) 
     
     
         33 . The method of  claim 27 , wherein assessing the oligosaccharide distribution of the recombinantly-expressed immunoglobulin comprises
 (a) assessing the level of agalactosyl fucosylated biantennary oligosaccharides (sum of NGA2F and NGA2F-GlcNac) present on the recombinantly-expressed immunoglobulin; and/or   (b) assessing the level of galactose containing fucosylated biantennary oligosaccharides (sum of NA1F and NA2F) present on the recombinantly-expressed immunoglobulin;   optionally, wherein the level of agalactosyl fucosylated biantennary oligosaccharides (sum of NGA2F and NGA2F-GlcNAc) present on the recombinantly-expressed 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 recombinantly-expressed immunoglobulin is 1%-30%, 2%-25%, 5%-20%, 5%-15%, 10%-20% or 27%-31%.   
     
     
         34 - 35 . (canceled) 
     
     
         36 . The method of  claim 27 , wherein the method is a fed batch process. 
     
     
         37 - 38 . (canceled)