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

Arthur Hinterholzer; Jennifer Moises; Christof Regl; Sebastian Schwap; Erdmann Rapp; Christian G Huber; Mario Schubert

doi:10.1080/19420862.2022.2132977

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

MAbs. 2022 Jan-Dec;14(1):2132977. doi: 10.1080/19420862.2022.2132977.

Authors

Arthur Hinterholzer^{1

2}, Jennifer Moises², Christof Regl^{1

2}, Sebastian Schwap^{2

3}, Erdmann Rapp^{4

5}, Christian G Huber^{1

2}, Mario Schubert^{1

2}

Affiliations

¹ Christian Doppler Laboratory for Innovative Tools for Biosimilar Characterization, University of Salzburg, Salzburg, Austria.
² Department of Biosciences and Medical Biology, University of Salzburg, Salzburg, Austria.
³ Bundesrealgymnasium Salzburg, Salzburg, Austria.
⁴ glyXera GmbH, Brenneckestraße, Magdeburg, Germany.
⁵ Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany.

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 ¹H-¹H TOCSY spectra followed by a confirmation using ¹H-¹³C 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.

Keywords: Galili epitope; NMR spectroscopy; monoclonal antibody; posttranslational modification; α-Gal.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Antibodies, Monoclonal*
Antineoplastic Agents, Immunological*
CHO Cells
Cricetinae
Cricetulus
Dithiothreitol
Epitopes
Galactose / chemistry
Magnetic Resonance Spectroscopy
Mice
Trisaccharides

Substances

Antibodies, Monoclonal
Antineoplastic Agents, Immunological
Epitopes
Trisaccharides
Dithiothreitol
Galactose

Grants and funding

The financial support from the Austrian Federal Ministry of Science, Research, and Economy, by a Start-up Grant of the State of Salzburg and the Austrian Research Promotion Agency (FFG) is gratefully acknowledged.