Methods for modulating protein glycosylation profiles of recombinant protein therapeutics using monosaccharides and oligosaccharides

Abstract

The present invention relates to the field of protein production, and in particular to methods and compositions for modulating glycosylation of proteins expressed in host cells.

We claim: 
     
         1 . A method of producing a composition comprising a protein with a modulated glycosylation profile, said method comprising:
 culturing a host cell expressing said protein in cell culture media supplemented with a monosaccharide and/or an oligosaccharide, thereby producing said composition comprising said protein with a modulated glycosylation profile as compared to a control,   wherein said control is a composition comprising a protein produced by culturing a host cell expressing said protein in cell culture media which is not supplemented with a monosaccharide and/or an oligosaccharide.   
     
     
         2 . The method of  claim 1 , further comprising purifying said composition comprising said protein with a modulated glycosylation profile. 
     
     
         3 . The method of  claim 1 , wherein the protein is an antibody or antigen-binding portion thereof. 
     
     
         4 . The method of  claim 3 , wherein the antibody is an anti-TNFα antibody. 
     
     
         5 . The method of  claim 4 , wherein the anti-TNFα antibody is adalimumab, or antigen binding fragment thereof. 
     
     
         6 . The method of  claim 1 , wherein the protein is a dual variable domain immunoglobulin (DVD-Ig). 
     
     
         7 . The method of  claim 1 , wherein the protein is selected from the group consisting of a TVD-IG, a half-body and a RAB. 
     
     
         8 . The method of  claim 1 , wherein the monosaccharide is tagatose. 
     
     
         9 . The method of  claim 1 , wherein the oligosaccharide is sucrose. 
     
     
         10 . The method of  claim 1 , wherein the cell culture media is supplemented with a sufficient amount of the monosaccharide to achieve a monosaccharide concentration selected from the group consisting of about 1 mM, about 5 mM, about 7 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM and about 100 mM. 
     
     
         11 . The method of  claim 10 , wherein the monosaccharide concentration is 30 mM. 
     
     
         12 . The method of  claim 10  or  11 , wherein the monosaccharide is tagatose. 
     
     
         13 . The method of  claim 1  wherein the cell culture media is supplemented with a sufficient amount of the oligosaccharide to achieve an oligosaccharide concentration selected from the group consisting of about 1 mM, about 5 mM, about 7 mM, about 10 mM, about 20 mM, about 30 mM. about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM and about 100 mM. 
     
     
         14 . The method of  claim 13 , wherein the oligosaccharide concentration is 30 mM. 
     
     
         15 . The method of  claim 13  or  14 , wherein the oligosaccharide is sucrose. 
     
     
         16 . The method of  claim 1 , wherein the modulated glycosylation profile of the protein comprises modulation of a fucosylation level and/or a mannosylated N-glycan oligosaccharide level in said protein. 
     
     
         17 . The method of  claim 16 , wherein the modulation of the fucosylation level comprises a decrease in the fucosylation level in said protein. 
     
     
         18 . The method of  claim 17 , wherein the decrease in the fucosylation level comprises a decrease in the level of NGA2F, NA1F-GlcNAc, NA1F and/or NA2F in said protein. 
     
     
         19 . The method of  claim 18 , wherein the decrease in the level of NGA2F, NA1F-GlcNAc, NA1F and/or NA2F is a decrease of about 0.1%, 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65%. 
     
     
         20 . The method of  claim 16 , wherein the modulation of the fucosylation level comprises an increase in the fucosylation level in said protein. 
     
     
         21 . The method of  claim 20 , wherein increase in the fucosylation level comprises an increase in the level of NGA2F-GlcNAc, NA1F-GlcNAc, NA1F and/or NA2F in said protein. 
     
     
         22 . The method of  claim 20 , wherein the increase in the level of NGA2F-GlcNAc, NA1F-GlcNAc, NA1F or NA2F is an increase of about 0.1%, 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15% or 20%. 
     
     
         23 . The method of  claim 17 , wherein the overall decrease in the fucosylation level comprises an increase or a decrease in the level of NGA2F, NGA2F-GlcNAc, NA1F-GlcNAc, NA1F and/or NA2F in said protein. 
     
     
         24 . The method of  claim 23 , wherein in the overall decrease in the fucosylation level is a decrease of about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%. 
     
     
         25 . The method of  claim 16 , wherein the modulation of the mannosylated N-glycan level comprises an increase in the mannosylation level of said protein. 
     
     
         26 . The method of  claim 25 , wherein the increase in the mannosylation level is an increase of about 0.1%, 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%. 
     
     
         27 . The method of  claim 25 , wherein the increase in the mannosylation level comprises an increase in the level of a high mannose N-glycan oligosaccharide selected from the group consisting of Man 5 glycan, Man 6 glycan, Man 7 glycan and Man 8 glycan. 
     
     
         28 . The method of  claim 27 , wherein the levels of Man 5 glycan, Man 6 glycan, Man 7 glycan and/or Man 8 glycan are increased by about 0.1%, 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40, 45% or 50%. 
     
     
         29 . The method of  claim 1 , wherein said host cell is a CHO cell. 
     
     
         30 . A method of producing a composition comprising an antibody, or antigen binding fragment thereof, with a modulated glycosylation profile, said method comprising:
 culturing a host cell expressing said antibody, or antigen binding fragment thereof, in cell culture media supplemented with sucrose and/or tagatose, thereby producing said composition comprising said antibody, or antigen binding fragment thereof, with an increased level of mannosylated N-glycans and a decreased level of fucosylated N-glycans as compared to a control,   wherein said control is a composition comprising an antibody, or antigen binding fragment thereof, produced by culturing a host cell expressing said antibody, or antigen binding fragment thereof, in cell culture media which is not supplemented with tagatose and/or glucose.   
     
     
         31 . The method of  claim 30 , wherein the antibody is adalimumab, or antigen binding fragment thereof. 
     
     
         32 . A method of producing a composition comprising an antibody, or antigen binding fragment thereof, with a modulated glycosylation profile, said method comprising:
 culturing a host cell expressing said antibody, or antigen binding fragment thereof, in cell culture media supplemented with sucrose and/or tagatose, thereby producing said composition comprising said antibody, or antigen binding fragment thereof, with a 1-50% increase in the level of mannosylated N-glycans and a 1-50% decrease in the level of fucosylated N-glycans as compared to a control,   wherein said control is a composition comprising an antibody, or antigen binding fragment thereof, produced by culturing a host cell expressing said antibody, or antigen binding fragment thereof, in cell culture media which is not supplemented with tagatose and/or glucose.   
     
     
         33 . The method of  claim 32 , wherein the antibody is adalimumab, or antigen binding fragment thereof. 
     
     
         34 . A composition comprising a cell culture media comprising a monosaccharide and/or an oligosaccharide. 
     
     
         35 . The composition of  claim 34 , wherein the monosaccharide is tagatose. 
     
     
         36 . The composition of  claim 34 , wherein the oligosaccharide is sucrose. 
     
     
         37 . A pharmaceutical composition comprising the composition produced by the methods of any one of  claims 1 ,  30  or  32  and a pharmaceutically acceptable carrier. 
     
     
         38 . A composition comprising a therapeutic protein with a modulated glycosylation profile produced by the methods of any one of  claims 1 ,  30  or  32 . 
     
     
         39 . The composition of  claim 38 , wherein the therapeutic protein is an antibody. 
     
     
         40 . A composition comprising a therapeutic protein, wherein said protein comprises a 1-50% increase in the level of mannosylated N-glycans and a 1-50% decrease in the level of fucosylated N-glycans, as compared to a control,
 wherein said control is a composition comprising a protein produced by culturing a host cell expressing said protein in cell culture media which is not supplemented with a monosaccharide and/or an oligosaccharide   
     
     
         41 . The composition of  claim 40 , wherein the therapeutic protein is selected from the group consisting of an antibody, an antigen-binding portion thereof, DVD-Ig, TVD-Ig, RAB and half-body. 
     
     
         42 . A composition of  claim 40 , wherein the therapeutic protein is an antibody. 
     
     
         43 . A method of producing a composition comprising an antibody, or antigen binding fragment thereof, with a modulated glycosylation profile, said method comprising:
 culturing a host cell expressing said antibody, or antigen binding fragment thereof, in cell culture media supplemented with sucrose and/or tagatose, thereby producing said composition comprising said antibody, or antigen binding fragment thereof, with a 1-30% increase in antibody-dependent cellular cytotoxicity (ADCC) response as compared to a control,   wherein said control is a composition comprising an antibody, or antigen binding fragment thereof, produced by culturing a host cell expressing said antibody, or antigen binding fragment thereof, in cell culture media which is not supplemented with tagatose and/or glucose.   
     
     
         44 . The method of  claim 43 , wherein the antibody is adalimumab, or antigen binding fragment thereof.