Methods of determining
glycan pairing content of a
protein composition are provided. In exemplary embodiments, the method comprises (a) treating a sample of the
protein composition with (i) a
mannosidase and (ii) a β1-4
endoglycosidase, to produce a mixture of components of the
protein, wherein at least some of the components comprises one or more digested
glycan structures; and (b) separating the components of the mixture based on molecular weight; and (c) quantifying the abundance of
glycan pairs, optionally, quantifying (i) paired afucosylated glycans, (ii) unpaired afucosylated glycans, (iii) paired
high mannose glycans, and / or (iv) unpaired
high mannose glycans, of the mixture. In exemplary aspects, the method comprises quantifying the relative unpaired afucosylated (AF) glycan content and / or relative unpaired
high mannose (HM) glycan content of the mixture.