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 .- 13 . (canceled) 
     
     
         14 . A method for detecting the presence or absence of fucose residues within a glycosylated antibody or a fragment thereof, comprising
 a) providing an antibody preparation comprising a glycosylated antibody or a fragment thereof,   b) removing heterogeneous saccharide residues from the antibody or the fragment thereof by enzymatic means,   c) removing other heterogeneous residues from the antibody or the fragment thereof by enzymatic means, and   d) analyzing the antibody or the fragment thereof for the presence or absence of fucose residues.   
     
     
         15 . The method of  claim 14 , further comprising a purification step prior to analyzing the antibody or the fragment thereof. 
     
     
         16 . The method of  claim 14 , further comprising determining the quantity of fucose residues and the distribution pattern of fucose residues among the molecules within the antibody preparation. 
     
     
         17 . The method of  claim 14 , further comprising determining the distribution of fucose residues per Fc molecule in the antibody preparation. 
     
     
         18 . The method of  claim 14 , wherein removing heterogeneous saccharide residues from the antibody or the fragment thereof is performed by one or more enzymes selected from the group consisting of Endo S and Endo H. 
     
     
         19 . The method of  claim 14 , wherein removing heterogeneous residues from the antibody or the fragment thereof is performed by one or more enzymes selected from the group consisting of plasmin and carboxypeptidase B. 
     
     
         20 . The method of  claim 14 , wherein analyzing the antibody or the fragment thereof is performed by an analysis selected from the group consisting of LC-MS analysis, CE-SDS MW analysis, and ESI-MS analysis. 
     
     
         21 . A method for detecting the presence or absence of fucose residues within a glycosylated antibody or a fragment thereof, comprising
 a) providing an antibody preparation comprising a glycosylated antibody,   b) optionally isolating the Fc fragment portion of the antibody preparation,   c) removing all heterogeneous saccharide residues from the glycosylated antibody or the Fc fragment with Endo H and Endo S,   d) removing C-terminal lysine residues from the antibody or the Fc fragment with carboxypeptidase B, and   e) analyzing the antibody or the Fc fragment for the presence or absence of fucose residues by LC-MS analysis, CE-SDS MW analysis, or ESI-MS analysis.   
     
     
         22 . The method of  claim 21 , further comprising a purification step prior to analyzing the antibody or the Fc fragment. 
     
     
         23 . The use of the method of  claim 14  or  claim 21  for determining cooperative fucosylation in an antibody preparation during fermentation. 
     
     
         24 . 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, instructions setting forth a procedure for the qualitative and quantitative detection of fucose residues within the peptide, and a container for the contents of the kit. 
     
     
         25 . The use of EndoS for cleaving complex-type oligosaccharides from a glycoprotein.