Methods of determining relative unpaired glycan content

Abstract

Methods of determining relative unpaired glycan content of an IgG antibody composition are provided. In exemplary embodiments, the method comprises treating the IgG antibody composition with two enzymes to forma mixture of Fab fragments and Fc fragments, wherein one enzyme cleaves an antibody heavy chain at a site N-terminal to the hinge region disulfide linkages and the other enzyme cleaves the β1,4 linkage between core GlcNAc residues to form Fc fragments each comprising a pair of core glycan structures, (b) separating the Fab fragments from the Fc fragments, and (c) quantifying the abundance of (i) paired afucosylated Fc fragments, (ii) unpaired afucosylated Fc fragments, (iii) paired high mannose Fc fragments, and/or (iv) unpaired high mannose Fc fragments, to determine the relative unpaired afucosylated (AF) glycan content and relative unpaired high mannose (HM) glycan content.

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