Methods to determine the level of afucosylation in recombinant monoclonal antibodies.
Glycosylation is a common posttranslational modification and a key quality attribute of recombinant monoclonal antibodies. In particular, the level of core-fucosylation is critical to several Fc mediated biological functions. However, it is challenging to accurately determine the level of afucosylation due to its low level and possible distribution in multiple oligosaccharide species. Methods that can accurately determine the level of afucosylation were developed in the current study. Digestion of monoclonal antibodies with Endoglycosidases F2 and H, which cleave the glycosidic bond between the two primary GlcNAc residues, reduced complicated oligosaccharide population into two groups with either only GlcNAc or GlcNAc and the core-fucose residue. Analysis of the digested antibodies by LC-MS provided a quick estimate of the level of afucosylation. Alternatively, PNGaseF released oligosaccharides were labeled with 2-aminobenzamide, digested with Endoglycosidases F2 and H followed by normal phase HPLC with fluorescence detection. The results demonstrated that the latter method can provide an accurate quantitation of the level of afucosylation.
Process Sciences Department, 100 Research Drive, Abbott Bioresearch Center, Worcester, Massachusetts 01605, USA.
Yang
Y
Shen
Hongcheng
H
Liu
D000116:Acetylgalactosamine / Q000737:chemistry / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005643:Fucose / Q000032:analysis*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D013058:Mass Spectrometry / Q000379:methods*; D010084:Oxidation-Reduction; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D062367:ortho-Aminobenzoates / Q000737:chemistry
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D062367:ortho-Aminobenzoates; D005643:Fucose; D006026:Glycoside Hydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D000116:Acetylgalactosamine; C000219:anthranilamide