A-fucosylation detection in antibodies

Abstract

This invention describes a new analytical method to determine the quantity and distribution of fucose per Fc within an antibody preparation.

1 . A method for detecting the presence or absence of fucose residues within a glycosylated antibody or a fragment thereof, comprising
 a) enzymatic removal of heterogeneous saccharide residues from the protein   b) enzymatic removal of other heterogeneous residues from the protein   c) subsequent analysis of the protein.   
     
     
         2 . The method of  claim 1 , wherein step c) additionally comprises a purification step prior to analysis. 
     
     
         3 . The method of  claim 1  or  2 , wherein the quantity of fucose residues and their distribution pattern among the molecules within an antibody preparation is determined. 
     
     
         4 . The method of any one of  claims 1  to  3 , wherein the distribution of fucose residues per Fc molecule in an antibody preparation is determined. 
     
     
         5 . The method of any one of  claims 1  to  4 , wherein the removal of step a) is performed by Endo S and/or Endo H. 
     
     
         6 . The method of any one of  claims 1  to  5 , wherein the removal of step b) is performed by plasmin and/or carboxypeptidase B. 
     
     
         7 . The method of any one of  claims 1  to  6 , wherein the analysis of step c) comprises LC-MS analysis, CE-SDS MW analysis or ESI-MS analysis. 
     
     
         8 . The method of any one of  claims 1  to  7 , comprising
 a) providing an antibody preparation 
 b) optionally isolating the Fc fragment portion of such antibody preparation 
 c) removing all heterogeneous saccharide residues from the antibody or Fc fragment with Endo H and Endo S 
 d) removing C-terminal lysine residues from the antibody or Fc fragment with carboxypeptidase B 
 e) analysis of the antibody or Fc fragment by LC-MS analysis, ESI-MS or CE-SDS MW analysis. 
 
     
     
         9 . The method of  claim 8 , wherein step e) additionally comprises a purification step prior to analysis. 
     
     
         10 . Use of the method of any one of  claims 1  to  9  for the determination of cooperative fucosylation in an antibody preparation during fermentation. 
     
     
         11 . A kit for use in qualitative and quantitative detection of fucose residues within a peptide, comprising Endo S and/or Endo H, plasmin and/or carboxypeptidase B, all components of the methods according to any one of  claims 1  to  9 , instructions setting forth a procedure according to any one of the methods of  claims 1  to  9 , and a container for the contents of the kit. 
     
     
         12 . Use of Endo S for cleavage of complex-type oligosaccharides from a glycoprotein. 
     
     
         13 . The methods, kits and uses substantially as hereinbefore described, especially with reference to the foregoing examples.