Native-MS Analysis of Monoclonal Antibody Conjugates by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.
Anal Chem
; 90(1): 745-751, 2018 01 02.
Article
em En
| MEDLINE
| ID: mdl-29193956
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 a monoclonal antibody (mAb) scaffold. Mass spectrometric (MS) characterization of these molecules affords highly accurate molecular weight (MW) and drug-to-antibody ratio (DAR) determination and is typically performed using orthogonal acceleration time-of-flight (oa-ToF) analyzers and more recently, Orbitrap instruments. Herein we describe for the first time the use of a 15 T 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 apodization 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 the 5 equivalent and 3.99 ± 0.09 versus 4.43 ± 0.01 for the 10 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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Espectrometria de Massas
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Biotina
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Imunoglobulina G
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Imunoconjugados
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Anticorpos Monoclonais
Idioma:
En
Revista:
Anal Chem
Ano de publicação:
2018
Tipo de documento:
Article