Native-MS analysis of monoclonal antibody conjugates by Fourier transform ion cyclotron resonance mass spectrometry

Campuzano, I.D.G., Netirojjanakul, C., Nshanian, M., Lippens, J.L., Kilgour, D.P.A. ORCID: 0000-0002-3860-7532, Van Orden, S.L. and Loo, J., 2017. Native-MS analysis of monoclonal antibody conjugates by Fourier transform ion cyclotron resonance mass spectrometry. Analytical Chemistry. ISSN 0003-2700

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Abstract

Antibody drug conjugates (ADCs) are an important class of therapeutic molecule currently being used to treat HER2-positive metastatic breast cancer, relapsed or refractory Hodgkin lymphoma, systemic anaplastic large cell lymphoma, relapsed or refractory B-cell precursor acute lymphoblastic leukemia and acute myeloid leukemia. An ADC typically consists of a small molecule or peptide-based cytotoxic moiety covalently linked, via lysine or cysteine residues, to amonoclonal antibody (mAb) scaffold. Mass spectrometric (MS) characterization of these molecules afford highly accurate molecular weight (MW) and drug-to-antibody ratio (DAR) determination, and is typically performed using orthogonal acceleration time-of-flight (oa-ToF) analysers and more recently Orbitrap instruments. Herein we describe for the first time the use of a 15 Tesla solariX Fourier transform ion cyclotron mass spectrometer to characterize an IgG1 mAb molecule conjugated with biotin via native lysine and cysteine residues, under native-MS and solution conditions. The cysteine biotin conjugates remained fully intact, demonstrating the ability of the FT-ICR to maintain the noncovalent interactions and efficiently transmit labile protein complexes. Native-MS was acquired and is displayed in magnitude mode using a symmetric Hann apodisation function. Baseline separation is achieved on all covalent biotin additions, for each charge state, for both the lysine and cysteine biotin-conjugates. Average DAR values obtained by native-MS for the lysine conjugate are compared to those derived by denaturing reversed phase liquid chromatography using an oa-ToF MS system (1.56 ±0.02 versus 2.24 ±0.02 for a 5-molar equivalent and 3.99 ±0.09 versus 4.43 ±0.01 for a 10-molar equivalent, respectively). Increased DAR value accuracy can be obtained for the higher biotin load, when using standard ESI conditions as opposed to nanoESI native-MS conditions. Both denatured LC-MS and native-MS spectral data were deconvoluted using a parsimonious based algorithm, without the need for parameter adjustment.

Item Type: Journal article
Publication Title: Analytical Chemistry
Creators: Campuzano, I.D.G., Netirojjanakul, C., Nshanian, M., Lippens, J.L., Kilgour, D.P.A., Van Orden, S.L. and Loo, J.
Publisher: American Chemical Society
Date: 1 December 2017
ISSN: 0003-2700
Identifiers:
NumberType
10.1021/acs.analchem.7b03021DOI
Divisions: Schools > School of Science and Technology
Depositing User: Jonathan Gallacher
Date Added: 06 Dec 2017 09:15
Last Modified: 06 Dec 2017 09:23
URI: http://irep.ntu.ac.uk/id/eprint/32146

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