Characterization of n-glycan mixtures by nuclear magnetic resonance

Abstract

The present disclosure provides nuclear magnetic resonance (NMR) methods for characterizing mixtures of N-linked glycans. Without limitation, methods of the present disclosure may be useful in characterizing monosaccharide composition, branching, fucosylation, sulfation, phosphorylation, sialylation linkages, presence of impurities and/or efficiency of a labeling procedure (e.g., labeling with a fluorophore such as 2-AB). In certain embodiments, the methods can be used quantitatively. In certain embodiments, the methods can be combined with enzymatic digestion to further characterize glycan mixtures.

1 - 91 . (canceled) 
     
     
         92 . A method of performing a quality control of a therapeutic glycoprotein preparation by NMR comprising steps of:
 obtaining a glycan preparation from a sample therapeutic glycoprotein preparation;   identifying whether an NMR spectrum for a sample of the glycan preparation includes an NMR signal that is associated with a structural characteristic of an N-glycan associated with a desired glycosylation pattern for a target therapeutic glycoprotein, wherein:   (i) the structural characteristic is an oligomannose structure and the step of identifying comprises determining whether the sample produces a  1 H signal with a chemical shift in the range of ca. 4.5 ppm to ca. 5.5 ppm;   (ii) the structural characteristic is a GlcNAc or sialic acid residue and the step of identifying comprises determining whether the sample produces a  1 H signal corresponding to an acetyl methyl nucleus of a GlcNAc or sialic acid residue;   (iii) the structural characteristic is a sialic acid residue and the step of identifying comprises determining whether the sample produces a  1 H signal corresponding to an axial or equatorial H3 nucleus of a sialic acid residue;   (iv) the structural characteristic is a sialic acid residue with an α2-3 linkage and the step of identifying comprises determining whether the sample produces a  1 H signal corresponding to an axial H3 nucleus of a sialic acid residue;   (v) the structural characteristic is a sialic acid residue with an α2-6 linkage and the step of identifying comprises determining whether the sample produces a  1 H signal corresponding to an axial H3 nucleus of a sialic acid residue;   (vi) the structural characteristic is di- or tri-acetylated NeuAc and the step of identifying comprises determining whether the sample produces a  1 H signal with a chemical shift at ca. 2.15 ppm;   (vii) the structural characteristic is a fucose residue and the step of identifying comprises determining whether the sample produces a  1 H signal corresponding to a methyl nucleus of a fucose residue;   (viii) the structural characteristic is a sialic acid and the step of identifying comprises determining whether the sample produces a  1 H- 1 H scalar correlation between the H3 axial and H3 equatorial nuclei of a sialic acid;   (ix) wherein the structural characteristic is a mono-antennary Man4 residue and the step of identifying comprises determining whether the sample produces a  1 H- 1 H scalar correlation between the H2 and H3 nuclei of a Man4 residue;   (x) the structural characteristic is a bi-antennary Man4 residue and the step of identifying comprises determining whether the sample produces a  1 H- 1 H scalar correlation between the H2 and H3 nuclei of a Man4 residue;   (xi) the structural characteristic is a galactose residue in a lactosamine extension and the step of identifying comprises determining the chemical shifts of a  1 H- 1 H scalar correlation between the H1 nucleus and another nucleus of a galactose residue;   (xii) the structural characteristic is a sulfated GlcNac residue and the step of identifying comprises determining the chemical shifts of a  1 H- 1 H scalar correlation between the H6 nucleus and another nucleus of a GlcNac residue;   (xii) the structural characteristic is a phosphorylated mannose residue and the step of identifying comprises determining the chemical shifts of a  1 H- 1 H scalar correlation between the H6 nucleus and another nucleus of a mannose residue;   (xiv) the structural characteristic is a core fucose residue and the step of identifying comprises determining the chemical shifts of a  1 H- 13 C scalar correlation for the anomeric nucleus of a GlcNAc2 residue;   (xv) the structural characteristic is a sialic acid residue and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to an axial H3 nucleus of a sialic acid residue;   (xvi) the structural characteristic is a sialic acid residue and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to an equatorial H3 nucleus of a sialic acid residue;   (xvii) the structural characteristic is an acetylated sialic acid residue and the step of identifying comprises determining the  1 H chemical shift of a  1 H- 13 C scalar correlation of the H7, H8 and/or H9 nuclei of a sialic acid residue;   (xviii) the structural characteristic is a Man4 residue and the step of identifying comprises determining the chemical shifts of a  1 H- 13 C scalar correlation corresponding to an anomeric nucleus of a Man4 residue;   (xix) the structural characteristic is a mono- or bi-antennary Man4 residue and the step of determining comprises determining whether the Man4 residue is mono-antennary or bi-antennary based on the chemical shifts of the  1 H- 13 C scalar correlation;   (xx) the structural characteristic is a Man4′ residue and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to an anomeric nucleus of a Man4′ residue;   (xxi) the structural characteristic is a mono- or bi-antennary Man4′ residue and the step of determining comprises determining whether the Man4′ residue is mono-antennary or bi-antennary based on the chemical shifts of the  1 H- 13 C scalar correlation;   (xxii) the structural characteristic is a GlcNAc1 residue and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to an anomeric nucleus of a GlcNAc1 residue;   (xxiii) the structural characteristic is a GlcNAc2 residue and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to an anomeric nucleus of a GlcNAc2 residue;   (xxiv) the structural characteristic is a Man3 residue and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to an anomeric nucleus of a Man3 residue;   (xxv) the structural characteristic is a GlcNac residue with a β(1-2) linkage to mannose and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to an anomeric nucleus of a GlcNac residue with a β(1-2) linkage to mannose;   (xxvi) the structural characteristic is a GlcNAc residue with a β(1-4) or β(1-6) linkage to mannose and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to an anomeric nucleus of a GlcNAc residue with a β(1-4) or β(1-6) linkage to mannose;   (xxvii) the structural characteristic is a GlcNAc residue in a lactosamine extension and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to an anomeric nucleus of a GlcNac residue in a lactosamine extension;   (xxviii) the structural characteristic is an unsubstituted galactose residue and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to an anomeric nucleus of an unsubstituted galactose residue;   (xxix) the structural characteristic is a galactose residue with an α(2-3) sialic acid attached and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to an anomeric nucleus of a galactose residue with an α(2-3) sialic acid attached;   (xxx) the structural characteristic is a galactose residue with an α(2-6) sialic acid attached and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to an anomeric nucleus of a galactose residue with an α(2-6) sialic acid attached;   (xxxi) the structural characteristic is a galactose residue in a lactosamine extension and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to an anomeric nucleus of a galactose residue in a lactosamine extension;   (xxxii) the structural characteristic is an oligomannose structure and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to an anomeric nucleus of an oligomannose structure;   (xxxiii) the structural characteristic is a core fucose residue and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to an anomeric nucleus of a core fucose residue;   (xxxiv) the structural characteristic is a core fucose residue and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to a methyl nucleus of a core fucose residue;   (xxxv) the structural characteristic is an antennary fucose residue and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to a methyl nucleus of an antennary fucose residue;   (xxxvi) the structural characteristic is a label attached to GlcNAc1 and the step of identifying comprises determining whether the sample produces a  1 H signal corresponding to GlcNAc1α H1;   (xxxvii) the structural characteristic is a label attached to GlcNAc1 and the step of identifying comprises determining whether the sample produces a split  1 H- 1 H scalar correlation corresponding to fucose H1/H2, H1/H3 or —CH 3 /H5 nuclei;   (xxxviii) the structural characteristic is a label attached to GlcNAc1 and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation corresponding to an anomeric nucleus of GlcNAc1α or GlcNAc1β;   (xxxix) the structural characteristic is a label attached to GlcNAc1 and the step of identifying comprises determining the chemical shifts of a  1 H- 13 C scalar correlation corresponding to an anomeric nucleus of a GlcNAc2 or Man3 residue;   (xxxx) the structural characteristic is a label attached to GlcNAc1 and the step of identifying comprises determining whether the sample produces a split  1 H- 13 C scalar correlation corresponding to an anomeric fucose nucleus; or   (xxxxi) the structural characteristic is a label attached to GlcNAc1 and the step of identifying comprises determining whether the sample produces a split  1 H- 13 C scalar correlation corresponding to a methyl fucose nucleus;   quantifying the NMR signal if the spectrum includes the signal;   quantifying the amount of N-glycans in the sample that have the structural characteristic; and   comparing the result of the quantifying the amount of N-glycans in the sample with a reference sample of the target therapeutic glycoprotein.   
     
     
         93 . The method of  claim 92 , wherein the identifying step comprises obtaining an NMR spectrum for the sample of the glycan preparation. 
     
     
         94 . The method of  claim 92 , further comprising recording the result of the comparing in a quality control record for the sample therapeutic glycoprotein preparation. 
     
     
         95 . The method of  claim 92 , wherein the target therapeutic glycoprotein is a therapeutic antibody and the sample therapeutic glycoprotein preparation is a sample therapeutic antibody preparation. 
     
     
         96 . The method of  claim 92 , wherein the target therapeutic glycoprotein is a therapeutic enzyme and the sample therapeutic glycoprotein preparation is a sample therapeutic enzyme preparation. 
     
     
         97 . The method of  claim 92 , wherein the target therapeutic glycoprotein is a therapeutic interferon and the sample therapeutic glycoprotein preparation is a sample therapeutic interferon preparation. 
     
     
         98 . The method of  claim 92 , wherein the target therapeutic glycoprotein is a therapeutic hematologic agent and the sample therapeutic glycoprotein preparation is a sample therapeutic hematologic agent preparation. 
     
     
         99 . The method of  claim 92 , wherein the target therapeutic glycoprotein is a therapeutic hormone and the sample therapeutic glycoprotein preparation is a sample therapeutic hormone preparation. 
     
     
         100 . The method of  claim 92 , wherein the target therapeutic glycoprotein is a therapeutic colony stimulating factor and the sample therapeutic glycoprotein preparation is a sample therapeutic colony stimulating factor preparation. 
     
     
         101 . The method of  claim 93 , wherein the step of obtaining an NMR spectrum comprises using a magnet having a strength of at least 600 MHz. 
     
     
         102 . The method of  claim 93 , further comprising a step of obtaining a signal integral within the NMR spectrum. 
     
     
         103 . The method of  claim 102 , wherein the signal integral is obtained by measuring signal intensity. 
     
     
         104 . The method of  claim 102 , wherein the signal integral is obtained by measuring signal area. 
     
     
         105 . The method of  claim 92 , wherein the sample therapeutic glycoprotein preparation comprises glycans in a state selected from the group consisting of free glycans, glycoconjugates, and cells. 
     
     
         106 . The method of  claim 92 , wherein the step of obtaining a glycan preparation comprises subjecting the sample therapeutic glycoprotein preparation to enzyme digestion so that glycans are released from glycoproteins in the glycoprotein preparation. 
     
     
         107 . The method of  claim 92 , wherein the step of obtaining a glycan preparation comprises obtaining a cell line that expresses a therapeutic glycoprotein of interest. 
     
     
         108 . The method of  claim 107 , wherein the therapeutic glycoprotein of interest is not naturally produced by the cell. 
     
     
         109 . The method of  claim 107 , wherein the comparing step comprises:
 comparing the NMR spectrum of the sample to an NMR spectrum of a reference sample, wherein the reference sample has an established glycosylation characteristic; and   based on the comparison, assessing the likelihood that the cells will generate the therapeutic glycoprotein of interest with a glycosylation characteristic close to the established glycosylation characteristic of the reference sample.   
     
     
         110 . The method of  claim 92 , wherein the step of identifying comprises obtaining one or more of a 2D NMR spectrum of the sample; a 2D  1 H- 1 H TOCSY NMR spectrum of the sample; a 1D selective  1 H TOCSY NMR spectrum of the sample; or a 2D  1 H- 13 C HSQC NMR spectrum of the sample. 
     
     
         111 . The method of  claim 92 , wherein the structural characteristic is a label attached to an N-glycan and the step of identifying comprises determining whether the sample produces a scalar correlation corresponding to said N-glycan. 
     
     
         112 . The method of  claim 92 , further comprising steps of treating the sample with a digestive enzyme to produce a digested sample and repeating the step of identifying with the digested sample. 
     
     
         113 . The method of  claim 92 , wherein the structural characteristic is an oligomannose structure and the step of identifying comprises determining whether the sample produces a  1 H signal with a chemical shift in the range of ca. 4.5 ppm to ca. 5.5 ppm. 
     
     
         114 . The method of  claim 92 , wherein the structural characteristic is a GlcNAc or sialic acid residue and the step of identifying comprises determining whether the sample produces a  1 H signal corresponding to an acetyl methyl nucleus of a GlcNAc or sialic acid residue. 
     
     
         115 . The method of  claim 92 , wherein the structural characteristic is a sialic acid residue and the step of identifying comprises determining whether the sample produces a  1 H signal corresponding to an axial or equatorial H3 nucleus of a sialic acid residue. 
     
     
         116 . The method of  claim 92 , wherein the structural characteristic is a sialic acid residue with an α2-3 linkage and the step of identifying comprises determining whether the sample produces a  1 H signal corresponding to an axial H3 nucleus of a sialic acid residue. 
     
     
         117 . The method of  claim 92 , wherein the structural characteristic is a sialic acid residue with an α2-6 linkage and the step of identifying comprises determining whether the sample produces a  1 H signal corresponding to an axial H3 nucleus of a sialic acid residue. 
     
     
         118 . The method of  claim 92 , wherein the structural characteristic is di- or tri-acetylated NeuAc and the step of identifying comprises determining whether the sample produces a  1 H signal with a chemical shift at ca. 2.15 ppm. 
     
     
         119 . The method of  claim 92 , wherein the structural characteristic is a fucose residue and the step of identifying comprises determining whether the sample produces a  1 H signal corresponding to a methyl nucleus of a fucose residue. 
     
     
         120 . The method of  claim 92 , wherein the structural characteristic is a sialic acid and the step of identifying comprises determining whether the sample produces a  1 H- 1 H scalar correlation between the H3 axial and H3 equatorial nuclei of a sialic acid. 
     
     
         121 . The method of  claim 92 , wherein the structural characteristic is a mono-antennary Man4 residue and the step of identifying comprises determining whether the sample produces a  1 H- 1 H scalar correlation between the H2 and H3 nuclei of a Man4 residue. 
     
     
         122 . The method of  claim 92 , wherein the structural characteristic is a bi-antennary Man4 residue and the step of identifying comprises determining whether the sample produces a  1 H- 1 H scalar correlation between the H2 and H3 nuclei of a Man4 residue. 
     
     
         123 . The method of  claim 92 , wherein the structural characteristic is a galactose residue in a lactosamine extension and the step of identifying comprises determining the chemical shifts of a  1 H- 1 H scalar correlation between the H1 nucleus and another nucleus of a galactose residue. 
     
     
         124 . The method of  claim 92 , wherein the structural characteristic is a sulfated GlcNac residue and the step of identifying comprises determining the chemical shifts of a  1 H- 1 H scalar correlation between the H6 nucleus and another nucleus of a GlcNac residue. 
     
     
         125 . The method of  claim 92 , wherein the structural characteristic is a phosphorylated mannose residue and the step of identifying comprises determining the chemical shifts of a  1 H- 1 H scalar correlation between the H6 nucleus and another nucleus of a mannose residue. 
     
     
         126 . The method of  claim 92 , wherein the structural characteristic is a core fucose residue and the step of identifying comprises determining the chemical shifts of a  1 H- 13 C scalar correlation for the anomeric nucleus of a GlcNAc2 residue. 
     
     
         127 . The method of  claim 92 , wherein the structural characteristic is a sialic acid residue and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to an axial H3 nucleus of a sialic acid residue. 
     
     
         128 . The method of  claim 92 , wherein the structural characteristic is a sialic acid residue and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to an equatorial H3 nucleus of a sialic acid residue. 
     
     
         129 . The method of  claim 92 , wherein the structural characteristic is an acetylated sialic acid residue and the step of identifying comprises determining the 1H chemical shift of a  1 H- 13 C scalar correlation of the H7, H8 and/or H9 nuclei of a sialic acid residue. 
     
     
         130 . The method of  claim 92 , wherein the structural characteristic is a Man4 residue and the step of identifying comprises determining the chemical shifts of a  1 H- 13 C scalar correlation corresponding to an anomeric nucleus of a Man4 residue. 
     
     
         131 . The method of  claim 92 , wherein the structural characteristic is a mono- or bi-antennary Man4 residue and the step of determining comprises determining whether the Man4 residue is mono-antennary or bi-antennary based on the chemical shifts of the  1 H- 13 C scalar correlation. 
     
     
         132 . The method of  claim 92 , wherein the structural characteristic is a Man4′ residue and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to an anomeric nucleus of a Man4′ residue. 
     
     
         133 . The method of  claim 92 , wherein the structural characteristic is a mono- or bi-antennary Man4′ residue and the step of determining comprises determining whether the Man4′ residue is mono-antennary or bi-antennary based on the chemical shifts of the  1 H- 13 C scalar correlation. 
     
     
         134 . The method of  claim 92 , wherein the structural characteristic is a GlcNAc1 residue and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to an anomeric nucleus of a GlcNAc1 residue. 
     
     
         135 . The method of  claim 92 , wherein the structural characteristic is a GlcNAc2 residue and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to an anomeric nucleus of a GlcNAc2 residue. 
     
     
         136 . The method of  claim 92 , wherein the structural characteristic is a Man3 residue and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to an anomeric nucleus of a Man3 residue. 
     
     
         137 . The method of  claim 92 , wherein the structural characteristic is a GlcNac residue with a β(1-2) linkage to mannose and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to an anomeric nucleus of a GlcNac residue with a β(1-2) linkage to mannose. 
     
     
         138 . The method of  claim 92 , wherein the structural characteristic is a GlcNAc residue with a β(1-4) or β(1-6) linkage to mannose and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to an anomeric nucleus of a GlcNAc residue with a β(1-4) or β(1-6) linkage to mannose. 
     
     
         139 . The method of  claim 92 , wherein the structural characteristic is a GlcNAc residue in a lactosamine extension and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to an anomeric nucleus of a GlcNac residue in a lactosamine extension. 
     
     
         140 . The method of  claim 92 , wherein the structural characteristic is an unsubstituted galactose residue and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to an anomeric nucleus of an unsubstituted galactose residue. 
     
     
         141 . The method of  claim 92 , wherein the structural characteristic is a galactose residue with an α(2-3) sialic acid attached and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to an anomeric nucleus of a galactose residue with an α(2-3) sialic acid attached. 
     
     
         142 . The method of  claim 92 , wherein the structural characteristic is a galactose residue with an α(2-6) sialic acid attached and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to an anomeric nucleus of a galactose residue with an α(2-6) sialic acid attached. 
     
     
         143 . The method of  claim 92 , wherein the structural characteristic is a galactose residue in a lactosamine extension and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to an anomeric nucleus of a galactose residue in a lactosamine extension. 
     
     
         144 . The method of  claim 92 , wherein the structural characteristic is an oligomannose structure and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to an anomeric nucleus of an oligomannose structure. 
     
     
         145 . The method of  claim 92 , wherein the structural characteristic is a core fucose residue and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to an anomeric nucleus of a core fucose residue. 
     
     
         146 . The method of  claim 92 , wherein the structural characteristic is a core fucose residue and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to a methyl nucleus of a core fucose residue. 
     
     
         147 . The method of  claim 92 , wherein the structural characteristic is an antennary fucose residue and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation with chemical shifts corresponding to a methyl nucleus of an antennary fucose residue. 
     
     
         148 . The method of  claim 92 , wherein the structural characteristic is a label attached to GlcNAc1 and the step of identifying comprises determining whether the sample produces a  1 H signal corresponding to GlcNAc1α H1. 
     
     
         149 . The method of  claim 92 , wherein the structural characteristic is a label attached to GlcNAc1 and the step of identifying comprises determining whether the sample produces a split  1 H- 1 H scalar correlation corresponding to fucose H1/H2, H1/H3 or —CH 3 /H5 nuclei. 
     
     
         150 . The method of  claim 92 , wherein the structural characteristic is a label attached to GlcNAc1 and the step of identifying comprises determining whether the sample produces a  1 H- 13 C scalar correlation corresponding to an anomeric nucleus of GlcNAc1α or GlcNAc1β. 
     
     
         151 . The method of  claim 92 , wherein the structural characteristic is a label attached to GlcNAc1 and the step of identifying comprises determining the chemical shifts of a  1 H- 13 C scalar correlation corresponding to an anomeric nucleus of a GlcNAc2 or Man3 residue. 
     
     
         152 . The method of  claim 92 , wherein the structural characteristic is a label attached to GlcNAc1 and the step of identifying comprises determining whether the sample produces a split  1 H- 13 C scalar correlation corresponding to an anomeric fucose nucleus. 
     
     
         153 . The method of  claim 92 , wherein the structural characteristic is a label attached to GlcNAc1 and the step of identifying comprises determining whether the sample produces a split  1 H- 13 C scalar correlation corresponding to a methyl fucose nucleus.