7
Table 1. Measured and theoretical masses for the mixture of eleven N-deglycosylated humanized antibodies at an
Orbitrap detection resolution of 140,000
Conclusion
In the analysis (0.3–5 min) using the Exactive Plus EMR
MS, molecular weight measurements of mAb and related
products in the low ppm mass deviation range allowed the
identification of all species simultaneously present in
solution. The number of DAR and relative abundance of
mAb/Ag complexes was also assessed with the peaks
intensities serving for relative quantification of the
detected species.
• The high resolving power of the Orbitrap mass analyzer
can baseline resolve a native mAb’s glycan peaks, as
well as the interference peaks, ensuring an excellent
mass accuracy in the low ppm range.
• The Exactive Plus EMR MS is able to sensitively
characterize ADC complexes with mass differences
between peaks corresponding to different additional
number of payloads/drugs. For each set of peaks, the
drug-to-antibody ratio (DAR) can be determined as
well as the relative ratio of each detected compound
in order to assess the mean DAR value.
• Native Orbitrap MS can reveal the number of antigens
bound to mAbs. Relative abundances of mAb/Ag
complexes at different stoichiometries can be achieved
from MS peak intesities.
• The Exactive Plus EMR MS enables the high through-
put screening of mAb mixtures, ensuring a excellent
mass accuracy for each individual mAb.
Species
Theoretical Masses
(Da)
Measured Masses
(Da)
Mass Accuracy
(ppm)
R
Rituximab
144186.3
144187.7
9.7
10
6F4-2 v10
144388.3
144387.5
5.5
9
6F4-2 v9
144420.5
144420.9
2.8
4
6F4-2 v 4
144498.4
144497.5
6.2
3
6F4-2 v3
144564.4
144564.6
1.4
6F4
6F4-2
144585.5
144590.9
37.3
7
6F4-2 v7
144732.5
144732.9
2.8
5
6F4-2 v5
144846.9
144846.5
2.8
1
6F4-2 v1
145015.3
145015.3
0
6
6F4-2 v6
145163.3
N.D
N.D
T
Trastuzumab
145165.5
145165.3
1.4
Acknowledgements
This work was in part supported by the OptimAbs
network bioclusters (LyonBiopole and Alsace Biovalley)
and sponsors (DGCIS, Oséo, Feder, Régions Rhône- Alpes
and Alsace, Communauté Urbaine de Strasbourg, CNRS,
University of Strasbourg).
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