Analytical Chemistry Chemical Cytometry Quantitates Superoxide

Figure 1. Illustration of the hinge region disulfide bonding pattern
of human (a) IgG2-A and (b) IgG2-B antibodies.
The ability to rapidly detect and characterize IgG2 isoforms is
of great interest, as it may help to facilitate the transition of new
IgG2 molecules from discovery into development and ultimately
commercialization. In recent years, mass spectrometry has played
an increasingly important role in the analytical characterization
of IgG therapeutics. Mass spectrometry is now widely used to
confirm the intact molecular weight of IgGs, 11,12 establish their
glycosylation profile, 13,14 and confirm 15 or establish 16 the primary
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6752 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
structure with a high degree of detail. Although MS is not
routinely used to characterize higher order structural elements
in IgGs, MS coupled with hydrogen/deuterium exchange was
recently demonstrated as a method to characterize the conformational
dynamics of IgG1 antibodies in solution. 17
Ion mobility mass spectrometry (IMMS) has shown great
promise as an intact protein separation and analysis methodology
to probe higher order structural elements including the overall
size/shapeofbiopolymersandlargemacromolecularassemblies. 18-28
Recently, Waters Corporation commercialized an ion mobility
mass spectrometer (Synapt) based on traveling waves (T-Wave). 29
In the T-Wave implementation of ion mobility, ion separation
occurs when a sequence of dc pulses push ions through the
mobility cell in the presence of an inert gas at relatively high
pressure. 29,30 The ability of an ion to “surf” the T-wave depends
on its collision cross section (CCS). Ions with compact structures
are pushed through the mobility cell faster than ions with more
elongated structures. In this work, we present evidence that
T-Wave IMMS can be used to separate disulfide variants of intact
IgG2 antibodies. Attractive features of the method include high
sensitivity (µg sample consumption), minimal sample preparation,
and fast analysis time (minutes).
EXPERIMENTAL SECTION
Human monoclonal antibodies mAb#1 (IgG2), mAb#2 (IgG1),
and mAb#3 (IgG2) were produced recombinantly in Chinese
hamster ovary (CHO) cells and purified at Amgen. All additional
reagents were purchased from Sigma-Aldrich (St. Louis, MO)
unless otherwise specified. For control experiments, constant
region two (CH2) domain N-glycans were removed by adding
1500 U of PNGase F (New England Biolabs, Ipswich, MA) per
100 µg of protein and incubating at 37 °C for 16 h.
Disulfide isoforms IgG2-A and IgG2-B were selectively enriched
using methods established by Dillon et al. 4 Briefly, to
enrich isoform B, IgG2s were incubated in 200 mM Tris buffer
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