Monoclonal Antibody and Related Product
Characterization Under Native Conditions
Using a Benchtop Mass Spectrometer
Yue Xuan
1
, François Debaene
2
, Johann Stojko
2
, Alain Beck
3
, Alain Van Dorsselaer
2
, Sarah Cianférani
2
and Maciej Bromiski
1
1
Thermo Fisher Scientific, Bremen, Germany
2
Institut Pluridisciplinaire Hubert Curien (IPHC), University of Strasbourg, Strasbourg, France
3
Centre d’Immunologie Pierre Fabre (CIPF), Saint Julien En Genevois, France
Application Note
597
Key Words
Orbitrap native MS, extended mass range MS, monoclonal antibody (mAb),
monoclonal antibody-drug-conjugate (ADC), monoclonal antibody antigen
complexes (mAb/Ag)
Goal
Demonstrate the characterization of mAbs, antibody-drug conjugates (ADC),
mAb/antigen (mAb/Ag) complexes, and a mixture of mAbs under their native
conditions by using a high-resolution, accurate-mass (HRAM) benchtop
mass spectrometer with extended mass range (EMR) in combination with a
chip-based electrospray ionization interface.
Introduction
Native mass spectrometry (MS) has emerged as a valuable
technique for characterization of intact noncovalent
protein complexes, reaching a high level of reliability
within the last ten years.
1
For the analysis of intact
monoclonal antibodies (mAbs), native MS yields accurate
mass measurements of the molecules, glycoform
identification, and assessment of higher-order structures
(dimer, trimer, tetramer), thus providing a robust, fast,
and reliable first-line analytical characterization tool.
2,3
This approach can now be applied to the routine
characterization of heterogeneous therapeutic monoclonal
antibodies. Native MS has gained interest not only for
analysis of intact mAb, but also for analysis of antibody-
drug conjugates (ADCs), bispecific mAbs, antibody-
antigen complexes, and characterization of antibody
mixtures. It benefits from simplified data interpretation
due to the presence of fewer charge states compared to
classical denaturing MS.
This application note describes the use of a new Orbitrap
mass spectrometer with an extended mass range of up to
m/z
20,000 and improved detection of high-mass ions for
the characterization of mAbs, ADCs, mAb/Ag, and mAb
mixtures under native conditions.
Experimental
Sample Preparation
The intact trastuzumab (Herceptin
®
, Roche), the monoclonal
antibody-drug conjugate brentuximab vedotin (ADC,
Adcetris
®
, Seattle Genetics), the mAb/antigen complexes
of J10.4 mAb/JAM-A, and one mixture of eleven distinct
IgG antibodies were introduced using the TriVersa
NanoMate
®
(Advion, USA) onto the Thermo Scientific
™
Exactive
™
Plus EMR Orbitrap
™
mass spectrometer.
Figure 1. Exactive Plus EMR mass spectrometer equipped
with a TriVersa NanoMate chip-based electrospray ionization
interface
