NATIVE ION MOBILITY MASS SPECTROMETRY FOR THE ...€¦ · resolution-IMS-MS (HR-IMS-MS) for...
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INTRODUCTION
Biopharmaceutical products provide a complex analytical
challenge to the modern pharmaceutical industry. Due to the
expansion of the biopharmaceutical industry and the
broadening range of drugs being investigated,
instrumentation is required to carry out advanced experiments
in a routine manner.
Over the past decade native ion mobility spectrometry-MS
(IMS-MS) has become commonplace in academic and
industrial research labs investigating protein folding1, protein-
ligand2 and protein-protein interactions, and protein complex
architecture3. However, more recently it is starting to gain
traction as an everyday tool.
Traditionally, native-IMS-MS is carried out using static
infusion from glass nanoflow capillaries following extensive
sample clean-up. However, with UPLC size-exclusion
chromatography (SEC) in a volatile, neutral pH mobile phase,
the benefits of native IMS-MS can be realized in a more
automated setting.
We describe a method to carry out native IMS-MS of antibody
therapeutics on a high resolution LC-IMS-MS platform. We
also describe investigations into the applicability of high
resolution-IMS-MS (HR-IMS-MS) for biopharmaceutical
analysis using a cyclic IMS-enabled research platform.
NATIVE ION MOBILITY MASS SPECTROMETRY FOR THE CHARACTERIZATION OF BIOTHERAPEUTICS
Dale A. Cooper-Shepherd, Jakub Ujma, Kevin Giles, Nick Tomczyk, Laetitia Denbigh Waters Corporation, Wilmslow, Cheshire, UK, SK9 4 AX
Figure 2. Schematic of the Vion IMS QTof.
METHODS
Size exclusion chromatography (SEC)-native IMS-MS was
conducted on trastuzumab (TmAb) and the antibody-drug
conjugate trastuzumab-emtansine (T-DM1, without
deglycosylation). Tmab and T-DM1 were reduced in the
presence of 5 mM dithiothreitol (DTT). Fragments were
released by reduction and incubation with IdeS enzyme
(Figure 1). SEC was conducted on an ACQUITY I-class UPLC
system with UPLC BEH200 SEC 2.1 x 150 mm, 1.7 µm
column. 100 mM ammonium acetate was used as the mobile
phase. SEC was conducted online with a Vion IMS QTof
(Waters Corp., Wilmslow, UK)(Figure 2). Source temperatures
and voltages were optimized for the transmission of intact
natively-folded protein ions. The acquisition mass range was
set to 8,000 m/z. Data were processed using UNIFI software.
The Vion IMS QTof applies the declustering (cone) voltage
after the IMS cell, which has the advantage of obtaining well-
desolvated protein ions whilst not disrupting native structure
and losing structural information.
References
1. T. W. Knapman, et al. Curr Anal Chem 2013, 9(2), 181-191.
2. T. M. Allison, et al. Nat Commun 2015; 6:8551
3. C. V. Robinson. Biochem Soc Trans 2017, 45(1):251-260.
RESULTS
SEC-Native MS spectra obtained from the Vion IMS QTof
were of high quality (Figure 4). TmAb showed excellent
separation of glycoforms, with peak widths in line with the
theoretical value. UNIFI data processing allows visualisation
of critical attributes (e.g. Glycoform ratio).
CONCLUSION
Native IMS-MS using SEC UPLC and the Vion IMS QTof yields high quality data for mAb and ADC characterization, including single-experiment determination of LC, HC, and intact mAb masses.
Workflows in UNIFI make repeat analyses streamlined for ease of comparison and data interpretation, e.g. DAR calculation.
IMS provides a means to compare 3D structure of ions in the gas phase.
A cyclic IMS-enabled research platform shows promise for the analysis of high MW proteins.
OVERVIEW
Here we present methods to characterize biotherapeutics
using native-ion mobility spectrometry-mass
spectrometry.
Size-exclusion chromatography coupled to the Vion
IMS QTof is a high performance solution for native-IMS
-MS analysis of biotherapeutics.
SEC-native IMS-MS has the potential to compare 3D
structures of antibody species.
Native MS under reducing conditions allows
determination of intact antibody and subunits in one
experiment.
A cyclic IMS-enabled research platform shows
promise for high molecular weight protein ions.
A)
B)
C)
Figure 5. TmAb structural differ-
ences under native conditions. A)
The ATD of non-reduced TmAb
shows two species, 1 and 2. Spe-
cies 1 is the major species and is
more compact than the minor spe-
cies 2 B) Under reducing condi-
tions species 2 contributes equally
to the intensity, indicating a greater
extent of unfolding in the structure.
A)
B)
Native IMS information is also obtained using the Vion IMS
QTof, allowing the determination of drift times for all ions.
SEC-Native-IMS was conducted on TmAb under standard
non-reducing and reducing conditions. Comparison of the drift
times of the intact native mAb under both conditions showed
significant differences, suggesting differences in their higher-
order structure (Figure 5). The reducing conditions have likely
affected the structure of TmAb by breaking a number of
disulphide bonds, which may cause the mAb to become less
rigid and exhibit a larger collision cross-section.
In addition to the intact mAb, native half-mAb and native light
chain (LC) were observed (Figure 6) under reducing
conditions, illustrating how the covalent structure of the
antibody is disrupted. Also, when elevated energies are used
both LC and HC product ions can be produced from the
native mAb. The light chain and heavy chain signals overlap
in m/z with the native light chain, but are well separated in
mobility meaning they do not interfere. These data show that
under reducing conditions it is possible to obtain the LC, HC,
and intact mAb masses in a single experiment.
A)
B)
Figure 6. Trastuzumab under partially-reducing conditions. A) Native
half-mAb, and light chain are observed as well as native intact mAb.
Elevated energies yield heavy chain and light chain as CID products. B)
UNIFI component plot showing masses of light chain, heavy chain, half-
mAb and intact mAb obtained from a single injection of trastuzumab un-
der partially-reducing conditions.
Figure 7. Calculating DAR of T-DM1 using native MS and UNIFI. A)
High quality native MS data were obtained without the need for deglyco-
sylation. B) MS data acquisition, deconvolution, mass identification and
DAR calculation are included in one method for swift results.
A)
B)
Enhanced resolution ion mobility experiments were performed
on a modified SYNAPT HDMS (ESI-Q-IMS-Tof) system fitted
with a prototype cyclic IMS device and dual gain ADC. Data
were processed in MassLynx and DriftScope software. The
cyclic IMS device and array (Figure 3) provide a 100cm,
single pass, mobility path length at 90° to the instrument axis
offering IMS resolution of 60-70 Ω/ΔΩ. By controlling the array
region ions can be made to do further passes increasing the
IMS path length by 1 meter per pass. In this way IMS
resolution in excess of 200 is possible. For more information
on this research platform please see poster TP385.
SEC-Native-MS was also conducted on an antibody-drug
conjugate trastuzumab-emtansine (T-DM1). The high quality
native MS data were acquired and automatically processed
using UNIFI software, including the calculation of drug-
antibody ratio (DAR) (Figure 7). As for TmAb, native IMS can
be conducted on T-DM1 under reducing conditions to obtain
masses of LC, HC and intact ADC (Figure 8).
Figure 8. T-DM1 under partially reducing conditions. Observed species
are analogous to those in Figure 6, with the addition of conjugations
originating from the ADC.
To investigate the high mass capability of our cyclic IMS-
enabled research platform, both intact deglycosylated T-DM1
and IdeS/reduced TmAb were prepared for static infusion
native IMS-MS.
Figure 3. Schematic of the cyclic IMS region of the research platform
Figure 9. Investigating the cyclic IMS-enabled research platform. A)
DAR 0 to DAR 4 ATDs from the 27+ charge state of T-DM1 after 2
passes. B) Mobility selection of regions of the ATD of the 13+ charge
state of the native Fc fragment of TmAb. After 4 passes around the de-
vice, mobility regions are selected and subjected to an additional pass.
Mobility-selected regions show different glycoform profiles due to mobil-
ity separation. Ion losses in the cell are minimal with additional passes.
A)
B)
Figure 1. Monoclonal antibody structure. The intact mAb can be decom-
posed into fragments. Using the IdeS enzyme and DTT reduction, the
light chain (LC), Fd’ and Fc fragments are produced. Using DTT alone,
the LC and heavy chain (HC) are produced.
Cyclic IMS of biotherapeutics - Preliminary studies
A)
B)
Figure 4. SEC-Native MS of TmAb. A) The SEC TIC trace is integrated
and combined to give the native mass spectrum at ~5500 m/z. B) Zoom
of the 27+ charge state of TmAb showing well separated glycoform
peaks. C) UNIFI component plot showing identified species from the de-
convoluted native mass spectrum.