Methods for modulating mannose content of recombinant proteins

Abstract

The present invention relates to methods of modulating (e.g., reducing) the mannose content, particularly high-mannose content of recombinant glycoproteins.

1 . A method of producing a composition of recombinant glycoprotein having low-mannose content comprising culturing a host-cell which expresses the recombinant glycoprotein in a culture medium having an osmolality of about 600 mOsm/Kg or less. 
     
     
         2 . A method of producing a composition of recombinant antibody, or an antigen-binding fragment thereof, having low-mannose content comprising culturing a host-cell which expresses the recombinant antibody or antigen-binding fragment thereof in a culture medium having an osmolality of about 600 mOsm/Kg or less. 
     
     
         3 . A method of producing a composition of recombinant human monoclonal antibody, or antigen-binding fragment thereof, that binds IL-15, comprising culturing a host-cell which expresses the antibody or antigen-binding fragment thereof in a culture medium having an osmolality of about 600 mOsm/Kg or less, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:4, or conservative amino acid substitutions thereof, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:2, or conservative amino acid substitutions thereof. 
     
     
         4 . The method of any of  claims 1  to  3 , wherein the osmolality of the culture medium is between about 250 and about 600 mOsm/Kg. 
     
     
         5 . The method of  claim 4 , wherein the osmolality of the culture medium is between about 250 and about 500 mOsm/Kg. 
     
     
         6 . The method of  claim 4 , wherein the osmolality of the culture medium is between about 250 and about 380 mOsm/Kg. 
     
     
         7 . The method of  claims 1  to  3 , wherein the culture medium comprises a salt selected from the group consisting of potassium at a concentration of about 70 mM or less, sodium at a concentration of about 200 mM or less, and combinations thereof. 
     
     
         8 . The method of  claim 7 , wherein the culture medium comprises a salt selected from the group consisting of
 (a) potassium at a concentration of about 10 mM to about 50 mM;   (b) sodium at a concentration of about 50 mM to about 100 mM; and   (c) combinations of (a) and (b).   
     
     
         9 . The method of  claims 1  to  3 , wherein the culture medium is substantially free of one or more amino acids selected from the group consisting of alanine, arginine, aspartic acid and glutamic acid. 
     
     
         10 . The method of  claims 1  to  3 , wherein the culture medium comprises one or more vitamins selected from the group consisting of biotin, D-calcium pantothenate, choline chloride, folic acid, i-inositol, niacinaminde, pyridoxal HCl, pyridoxine HCl, riboflavin, thiamine HCl and cyanocobalamin, at a concentration of about 0.00005 g/L to about 0.9 g/L. 
     
     
         11 . The method of  claims 1  to  3 , wherein the culture medium comprises glucose at a concentration of about 1 mM to about 90 mM. 
     
     
         12 . The method of  claims 1  to  3 , wherein the culture medium comprises one or more peptones selected from the group consisting of yeast extract, yeast hydrolysate, soy peptone, soy hydrolysate, wheat peptone and wheat hydrolysate, at a concentration of about 0.5 g/L to about 60 g/L. 
     
     
         13 . The method of  claims 1  to  3 , wherein the culture medium comprises at least one osmo-protectant in an amount necessary to maintain the osmolality at about 600 mOsm/Kg or less. 
     
     
         14 . The method of  claim 13 , wherein the osmo-protectant is selected from the group consisting of betaine, glycine, L-threonine, L-proline, and derivatives thereof. 
     
     
         15 . The method of  claim 13 , wherein the osmo-protectant is betaine at a concentration of about 1 mM to about 100 mM, 
     
     
         16 . The method of  claim 15 , wherein the betaine is present at a concentration from about 20 mM to about 30 mM. 
     
     
         17 . The method of any of  claims 1  to  3 , wherein the host-cell is cultured for a period of about 5 to about 14 days. 
     
     
         18 . The method of any of  claims 1  to  3 , wherein the host-cell is cultured at a temperature of about 31° C. to about 38° C. 
     
     
         19 . The method of any of  claims 1  to  3 , wherein the host cell is a mammalian cell. 
     
     
         20 . The method of  claim 19 , wherein the mammalian host cell is a CHO cell. 
     
     
         21 . A composition of recombinant glycoprotein produced by the method of  claim 1 . 
     
     
         22 . A composition of recombinant antibody or antigen-binding fragment thereof produced by the method of  claim 2 . 
     
     
         23 . A composition of human monoclonal antibody or an antigen-binding fragment thereof that binds IL-15, produced by the method of  claim 3 . 
     
     
         24 . A composition comprising an isolated antibody or an antigen-binding fragment thereof having low-mannose content. 
     
     
         25 . A composition comprising an isolated human monoclonal antibody that binds IL-15, or an antigen-binding fragment thereof, having low-mannose content. 
     
     
         26 . A composition of a human monoclonal antibody having low-mannose content, said antibody comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:4, or an amino acid sequence of at least 90% or at least 95% identity thereto, or conservative amino acid substitutions thereof, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:2, or an amino acid sequence of at least 90% or at least 95% identity thereto, or conservative amino acid substitutions thereof. 
     
     
         27 . A composition of a human monoclonal antibody having low-mannose content, said antibody comprising a light chain variable region comprising one or more CDRs comprising amino acid sequences set forth in SEQ ID NOs:8-10, or an amino acid sequence of at least 90% or at least 95% identity thereto, or conservative amino acid substitutions thereof, and a heavy chain variable region comprising one or more CDRs comprising amino acid sequences set forth in SEQ ID NOs:5-7, or an amino acid sequence of at least 90% or at least 95% identity thereto, or conservative amino acid substitutions thereof. 
     
     
         28 . The composition of  claim 25 , further comprising a pharmaceutically acceptable carrier. 
     
     
         29 . A method of treating or preventing a disorder that is associated with overexpression of human IL-15 and/or in which a downregulation or inhibition of human IL-15 induced effects is beneficial, comprising administering to a subject an isolated antibody produced by the method of  claim 3 . 
     
     
         30 . The method of  claim 29 , wherein said disorder is selected from the group consisting of: vasculiitis, psoriasis, multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, allograft rejection, graft versus host disease, T-cell lymphoma, and T-cell leukemia. 
     
     
         31 . The method of  claim 30 , wherein said disorder is an inflammatory bowel disease. 
     
     
         32 . The method of  claim 31 , wherein said inflammatory bowel disease is Crohn's disease or celiac disease. 
     
     
         33 . The method of  claim 29 , wherein the disorder is selected from the group consisting of arthritides, connective tissue disorders, ophthalmological disorders, neurological disorders, gastrointestinal and hepatic disorders, allergic disorders, hematologic disorders, skin disorders, pulmonary disorders, malignancies, transplantation-derived disorders, endocrinologic disorders, vascular disorders, gynecological disorders and infectious diseases. 
     
     
         34 . A composition of  claim 26  or  27 , wherein no more than about 10% of the antibodies in said composition comprises M5 or larger. 
     
     
         35 . A composition of  claim 26  or  27 , wherein no more than about 5% of the antibodies in said composition comprises M5 or larger. 
     
     
         36 . A composition of  claims 26  or  27 , wherein about 4% of antibodies in said composition comprises M5 or greater. 
     
     
         37 . A method of detecting and/or quantitating the high-mannose content of a glycoprotein in a sample, said method comprising subjecting said sample to an endoglycosidase digestion followed by separating the digested glycoproteins by electrophoresis. 
     
     
         38 . The method of  claim 37 , wherein the endoglycosidase comprises Endoglycosidase H and the digestion is carried out at 37° C. for about 2 hours. 
     
     
         39 . The method of  claim 37 , wherein the electrophoreis comprises denaturing capillary electrophoresis. 
     
     
         40 . The method of  claim 37 , wherein prior to the separating step the digested sample is reduced using a reducing agent 
     
     
         41 . The method of  claim 40 , wherein the reducing agent comprises β-mercaptoethanol. 
     
     
         42 . The method of  claim 37 , wherein the glycoprotein comprises an antibody produced from a hybridoma or an eukaryotic host cell.