Gal alpha 1-3gal-containing n-glycans in glycoprotein products derived from cho cells

Abstract

The present invention provides methods of evaluating CHO cells.

1 - 50 . (canceled) 
     
     
         51 . A method of producing a therapeutic glycoprotein, comprising:
 (a) culturing CHO cells under conditions suitable for expression of a recombinant glycoprotein comprising one or more glycans, wherein the CHO cells have not been genetically engineered to produce terminal alpha-galactosyl residues on glycans;   (b) treating the one or more glycans of the recombinant glycoprotein with one or more exoglycosidases;   (c) detecting digested terminal galactose-alpha-1-3-galactose residues to thereby measure glycans containing terminal galactose-alpha-1-3-galactose residues produced by the CHO cells; and   (d) producing a therapeutic glycoprotein in the CHO cells if a target level of terminal galactose-alpha-1-3-galactose residues is measured.   
     
     
         52 . The method of  claim 51 , wherein the CHO cells are in a cell culture. 
     
     
         53 . The method of  claim 51 , wherein the detecting is performed on any of: the recombinant glycoprotein isolated from the CHO cells, peptides obtained from the recombinant glycoprotein expressed by the CHO cells, cell surface glycans of the CHO cells, glycan preparations obtained from the CHO cells, glycan preparations obtained from the recombinant glycoprotein expressed by the CHO cells, and combinations thereof. 
     
     
         54 . The method of  claim 51 , further comprising isolating the recombinant glycoprotein from the CHO cells before step (b). 
     
     
         55 . The method of  claim 52 , wherein the cell culture is in a bioreactor. 
     
     
         56 . The method of  claim 51 , wherein the detecting step comprises use of a technique selected from the group consisting of: chromatographic methods, mass spectrometry (MS) methods, electrophoretic methods, nuclear magnetic resonance (NMR) methods, monosaccharide analysis, fluorescence methods, UV-VIS absorbance, enzymatic methods, and combinations thereof. 
     
     
         57 . The method of  claim 51 , wherein the therapeutic glycoprotein is a human therapeutic glycoprotein, and in step (d) the CHO cells have been transformed with a vector encoding the human therapeutic glycoprotein. 
     
     
         58 . The method of  claim 51 , wherein the CHO cells are from at least one of: at least two different CHO strains, at least two different clonal cell populations, and at least two different samples from a cell culture in a manufacturing process train for a therapeutic glycoprotein. 
     
     
         59 . The method of  claim 51 , wherein the CHO cells are from two or more CHO cell populations and the method further comprises comparing the levels of glycans containing terminal galactose-alpha-1-3-galactose residues produced by the CHO cells of the two or more CHO cell populations. 
     
     
         60 . The method of  claim 51 , further comprising comparing the level of glycans containing terminal galactose-alpha-1-3-galactose residues produced by the CHO cells to a reference glycoprotein sample. 
     
     
         61 . The method of  claim 51 , further comprising recording the measured level of glycans containing terminal galactose-alpha-1-3-galactose residues in a print or computer-readable medium. 
     
     
         62 . The method of  claim 51 , wherein the target level is a quality criterion for a pharmaceutical preparation. 
     
     
         63 . The method of  claim 51 , wherein the target level is a range or value in a product specification. 
     
     
         64 . The method of  claim 51 , wherein the target level is no more than 5% terminal galactose-alpha-1-3-galactose. 
     
     
         65 . The method of  claim 51 , wherein detecting comprises use of a detection molecule. 
     
     
         66 . The method of  claim 51 , wherein the one or more exoglycosidases are selected from the group consisting of sialidase, galactosidase, hexosaminidase, mannosidase, and fucosidase. 
     
     
         67 . The method of  claim 51 , wherein the treating step comprises treating with one or more exoglycosidases for a time and under conditions suitable for the one or more exoglycosidases to cleave one or more terminal glycosidic bonds from a non-reducing end of the one or more glycans. 
     
     
         68 . A method of monitoring CHO cells for production of a target recombinant glycoprotein comprising glycans containing terminal galactose-alpha-1-3-galactose epitopes, the method comprising:
 (a) producing a target recombinant glycoprotein comprising one or more glycans by culturing CHO cells under conditions suitable for expression of the target recombinant glycoprotein by the CHO cells, wherein the CHO cells have not been genetically engineered to produce terminal alpha-galactosyl residues on glycans;   (b) removing at least first and second glycan-containing samples of the target recombinant glycoprotein from the culture;   (c) treating the first and second glycan-containing samples with one or more exoglycosidases; and   (d) detecting digested terminal galactose-alpha-1-3-galactose residues in the first and second glycan-containing samples to thereby measure glycans containing terminal galactose-alpha-1-3-galactose residues in the first and second glycan-containing samples of the target recombinant glycoprotein.   
     
     
         69 . The method of  claim 68 , wherein the first and second glycan-containing samples are removed from the culture at different times. 
     
     
         70 . The method of  claim 68 , further comprising comparing the measured glycans containing terminal galactose-alpha-1-3-galactose residues in the first and second glycan-containing samples of the target recombinant glycoprotein. 
     
     
         71 . A method of producing a therapeutic glycoprotein, comprising:
 (a) culturing CHO cells under conditions suitable for expression of a recombinant glycoprotein comprising one or more glycans, wherein the CHO cells have not been genetically engineered to produce terminal alpha-galactosyl residues on glycans;   (b) measuring glycans containing terminal galactose-alpha-1-3-galactose residues on the recombinant glycoprotein by NMR; and   (c) producing a therapeutic glycoprotein in the CHO cells if a target level of terminal galactose-alpha-1-3-galactose residues is measured.   
     
     
         72 . A method of monitoring CHO cells for production of a target recombinant glycoprotein comprising glycans containing terminal galactose-alpha-1-3-galactose epitopes, the method comprising:
 (a) producing a target recombinant glycoprotein comprising one or more glycans by culturing CHO cells under conditions suitable for expression of the target recombinant glycoprotein by the CHO cells, wherein the CHO cells have not been genetically engineered to produce terminal alpha-galactosyl residues on glycans;   (b) removing at least first and second glycan-containing samples of the target recombinant glycoprotein from the culture; and   (c) measuring glycans containing terminal galactose-alpha-1-3-galactose residues in the first and second glycan-containing samples of the target recombinant glycoprotein by NMR.