Chemical Structure and Composition of Major Glycans Covalently Linked to Therapeutic Monoclonal Antibodies by Middle-Down Nuclear Magnetic Resonance

Abstract

Glycosylation of monoclonal antibodies (mAbs) is a critical quality attribute that can impact mAb drug efficacy and safety. The mAb glycans are inherently heterogeneous in chemical structure and composition of monosaccharides. The established fluorescence or mass-spectrometry (MS) detection methods for glycosylation evaluation may require multiple steps of glycan cleavage or extensive digestion of the mAb, chemical labeling of the glycans, column separation and report the chemical identity of glycans indirectly through retention time and molecular weight values. In demonstrating chemical structure similarity and comparability among mAb drugs, orthogonal analytical methods for measuring glycan chemistry are needed to ensure the quality of drug products. Here, a "middle-down" NMR method is developed as a proof-of-concept approach to measure the domain-specific glycosylation of marketed mAb drugs without cleavage of the glycan moieties. Complete glycan ¹H/¹³C chemical shift assignments were obtained at ¹³C natural abundance from commercial standard glycans that allowed unambiguous determination of the chemical structure, glycosidic linkage position, and anomeric configuration of each monosaccharide in the major N-glycan scaffolds found in mAb molecules. The analysis of glycan anomeric peaks in two-dimensional (2D) ¹H-¹³C NMR spectra yielded metrics for clinically important mAb quality attributes (i.e., galactosylation (Gal%) and fucosylation (Fuc%)), consistent with literature results using a standard glycan-mapping method. Therefore, the middle-down NMR method provided a facile orthogonal measurement for mAb glycosylation characterization with improved chemical information content on glycan structure determination and quantification, compared to standard approaches.

Figure 1.

Structure illustrations for standard glycans. (a) Molecular structure of G2F. For GN1, the β configuration was drawn, but both α and β configurations are in equilibrium for free glycan. Man4′, GN5′, and Gal6′ represent the α1–α6 branched saccharides from Man3. (b) Symbolic representation of common glycans found in mAbs.

Figure 2.

Full (left) and the expanded glycan region (right) of 2D ¹H–¹³C HSQC spectrum of rituximab from Rituxan. Blue box indicates glycan signal range; red box indicates fingerprint anomeric signals from glycans.

Figure 3.

Fingerprint anomeric regions of HSQC spectra of all studied mAb drugs. All spectra, except the bottom right one, were obtained from the Fc domains of the mAbs. The plotting threshold of the signal-to-noise (S/N) ratio was 10 for all spectra. GN5/5′a indicated galactosylated GN5/5′, while GN5/5′b indicated the terminal GN5/5′. The crosspeaks a, c, and d were respectively assigned to the anomeric peaks of ManB, Man4′, and ManA of the high mannose glycans MAN-5 and MAN-6. The crosspeaks e and f were anomeric peaks of Man4 of the high mannose MAN-5 and MAN-6, respectively. The crosspeaks b, g, h, and i were not assigned.