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 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
• α-Gal
• NMR spectroscopy
• monoclonal antibody
• posttranslational modification

Acknowledgments

We acknowledge Novartis for kindly providing expired samples of reference medicines for rituximab, adalimumab and abatacept, and Dr Urs Lohrig and Dr Chiara Cabrele for valuable comments on the manuscript as well as scientific discussions. We also acknowledge Biomolecular NMR Spectroscopy Platform at the ETH Zürich, Dr Alvar Gossert, and Dr Simon Rüdisser (both ETH Zürich) for access to 500 MHz and 700 MHz Bruker spectrometer with cryogenic probe and technical support.

Disclosure statement

The authors declare the following competing financial interest(s): The salary of Arthur Hinterholzer was fully funded and Christian G. Hubers salary is partly funded by the Christian Doppler Laboratory for Biosimilar Characterization, which is partly supported by Novartis and Thermo Fisher Scientific. Erdmann Rapp is an employee of glyXera GmbH, Magdeburg, Germany. The authors declare no other competing financial interest.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19420862.2022.2132977

Additional information

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.