Unambiguous identification of α-Gal epitopes in intact monoclonal antibodies by NMR spectroscopy

Abstract

The α-Gal epitope consisting of the terminal trisaccharide Galα1,3Galβ1,4GlcNAc exposed on cell or protein surfaces can cause severe immune reactions, such as hypersensitivity reactions, in humans. This epitope is also called the xenotransplantation epitope because it is one of the main reasons for the rejection of non-human organ transplants by the human innate immune response. Recombinant therapeutic proteins expressed in murine cell lines may contain α-Gal epitopes, and therefore their absence or presence needs to be tightly monitored to minimize any undesired adverse effects. The analytical identification of α-Gal epitopes in glycoproteins using the common standard techniques based on liquid chromatography and mass spectrometry is challenging, mainly due to the isobaricity of hexose stereoisomers. Here, we present a straightforward NMR approach to detect the presence of α-Gal in biotherapeutics based on a quick screen with sensitive 1 H- 1 H TOCSY spectra followed by a confirmation using 1 H- 13 C HSQC spectra. Abbreviations : α-Gal: α1,3-linked galactose; AGC: automatic gain control; CHO: Chinese hamster ovary; CE: capillary electrophoreses coupled to mass spectrometry; COSY: correlation spectroscopy; DSS: 2,2-dimethyl-2-silapentane-5-sulfonate; DTT: dithiothreitol; GlcNAc: N-acetyl glusomamine; HCD: higher-energy collisional dissociation; HMBC: heteronuclear multiple-bond correlation; HPLC: high-performance liquid chromatography; HSQC: heteronuclear single-quantum corre; LacNAc: N-acetyl lactosamine; mAb: monoclonal antibody; MS: mass spectrometry; NMR: nuclear magnetic resonance; NOESY: 2D) nuclear Overhauser spectroscopy; PEG: polyethylenglycol; pH*: observed pH meter reading without correction for isotope effects; PTM: post-translational modification; TCEP: tris(2-carboxyethyl) phosphine hydrochloride; TOCSY: total correlation spectroscopy; xCGE-LIF: multiplex capillary gel electrophoresis with laser-induced fluorescence detection.

The NMR signature of maltose-based glycation in full-length proteins

“…1 h depending on the recycle delay and the resolution chosen in the indirect dimension. It is not so common to have isolated and characteristic signals of PTMs in 1 H- 1 H TOCSY spectra, because dispersion in 1 H is much smaller than in 13 C, but rare cases like characteristic signals of α-Gal epitopes (Hinterholzer et al 2022 ) and now maltose-glycation allow the unambiguous detection in a time efficient and easy to interpret manner.…”

“…The sample preparation was previously described (Hinterholzer et al 2022 ). In brief, abatacept (ORENCIA®, Bristol Myers Squibb; Lot.…”

“…Using deuterated urea in D 2 O, in which the lyophilized protein of interest is dissolved, and adding reducing agent for eliminating disulfide bonds results in simplified spectra showing signals with random coil chemical shifts. Ideally, each PTM is recognized by a unique chemical shift correlation pattern that is sufficiently different from random coil correlations of the proteinogenic amino acids within a protein as illustrated for such diverse modifications as specific glyco epitopes (Unione et al 2019 ; Schubert et al 2015 ; Hinterholzer et al 2022 ; Hargett et al 2021 ; Peng et al 2018 ), oxidation products (Hinterholzer et al 2020 ), deamidation products (Grassi et al 2017 ), pyroglutamate (Hinterholzer et al 2019 ), aspartate isomerization (Hinterholzer et al 2021 ) and glycation (Moises et al 2022 ). Typically the observed patterns are so characteristic that an unambiguous identification of a certain modification is achieved, which is very reliable and orthogonal to HPLC-MS 2 techniques.…”

Unambiguous Identification of Glucose-Induced Glycation in mAbs and other Proteins by NMR Spectroscopy

“…Ideally, each PTM leads to a characteristic chemical shift correlation pattern that is distinguishable from random coil correlations of the proteinogenic amino acids within a protein. PTMs with a characterized NMR signature include pyroglutamate formation [ 9 ], oxidation [ 8 ], deamidation of Asn and strand cleavage after Asp [ 7 , 10 ], terminal ends of glycans including high-mannose and certain types of complex glycans [ 11 , 12 , 17 , 19 ] and the immunogenic α-Gal epitope [ 20 ].…”