6
Native MS Analysis of a Mixture of Eleven
N-deglycosylated Humanized Antibodies
Analysis of mAb mixtures is of utmost interest for
high-throughput screening purposes and for therapeutic
use to block simultaneously multiple epitopes. Indeed
cocktails of mAbs with additive or synergic effects are
increasingly foreseen as potential new therapeutic entities.
Figure 5A presents a convoluted mass spectrum of a mixture
of eleven distinct deglycosylated humanized IgG antibodies.
This mix includes two marketed therapeutic mAbs
(rituximab and trastuzumab) and nine point mutation
variants of the Hz6F4-2 mAb.
4,5
Figure 5B shows a full
native mass spectrum of the mAb mix with an in-source
CID energy set to 100 eV.
The well-resolved ion signals at a detection resolution of
140,000 and accurately measured masses enable the
unambiguous assignment of ten out of the eleven compounds.
Trastuzumab and Hz6F4-2v6 could not be differentiated due
to very close molecular weights (2 Da). Peaks corresponding
to Hz6F4-2 and Hz6F4-2v3, which differ by only 21 Da in
mass, are clearly distinguished on the mass spectrum.
However, they are not baseline resolved, and when combined
with the low signal-to-noise (S/N) ratio (S/N < 20), that
causes a relatively low mass accuracy for Hz6F4-2. However,
with a good signal-to-noise ratio (S/N > 50), even without
baseline-resolved peaks, for example, peaks of Hz6F4-2v9
and 6F4-2v10, the mass accuracies are achieved in the low
ppm range for both species. The measured and theoretical
masses for the mixture of eleven N-deglycoslated humanized
antibodies are listed in Table 1.
Figure 5. Native MS analysis of a mixture of eleven N-deglycosylated humanized antibodies. A. Deconvoluted mass spectrum. B. Charge
state distribution in native conditions.
3000
4000
5000
6000
7000
8000
9000
10000
m/z
5807.5
6049.3
6305.8
5584.4
5377.4
6592.5
5185.7
5006.5
6914.1
A
B
