1
[ APPLICATION NOTE ]
TOF MS
WATERS SOLUTIONS
BioAccord™ System (ACQUITY™ UPLC™
I-Class PLUS and RDa Detector)
IonHance™ CX-MS pH Concentrates
UNIFI™ Scientific Information System
KEYWORDS
IEX-MS, charge variants, MS-compatible,
BioResolve SCX mAb, IonHance
CX-MS pH Concentrates
APPLICATION BENEFITS
■
■
A novel salt mediated pH gradient ion
exchange (IEX) method is demonstrated
that employs volatile salts to enable
direct coupling of mass spectrometry.
■
■
The ability to directly couple IEX-MS
reduces the dependency on traditional
fractionation methods by facilitating
the direct and simple identification
of chromatographic peaks.
INTRODUCTION
Characterization of charge heterogeneity is critical for the development of
biotherapeutic drugs, as many of these charge variants can have an impact
on drug potency and efficacy.1,2 Therefore, it is important to understand the
possible impacts of charge variants and to monitor them throughout discovery,
development, and manufacturing. Regarding charge variant characterization,
options for analytical techniques include ion-exchange chromatography
(IEX) or methods of capillary electrophoresis (CE) such as capillary zone
electrophoresis (CZE) or isoelectric focusing (IEF). While all these methods
are used to some degree for the analysis of charge variant heterogeneity,
there are certain advantages and disadvantages to each of them.
The advantages of CE-based methods include less risk of non-specific
interactions as there is no stationary phase3,4 and increasing feasibility to
couple to mass spectrometry (MS). The disadvantages of CE include the
limitation in sample loading and poor reproducibility, both of which can
complicate or limit fraction collection capabilities.5,6 IEX, on the other hand,
offers chromatographic reproducibility and considerably higher sample
loading capacity. However, traditionally, IEX separations require high
concentrations of salts that are not compatible with mass spectrometry
(MS) analysis, which has left a gap in the characterization of charge variants.
Recently, it has been shown7,8 that direct IEX-MS characterization of these
charge variants is possible, if volatile salts are employed. Here we present a
novel, direct IEX-MS method using ammonium-based mobile phases which
is applicable to a wide range of monoclonal antibody (mAb) species. The
analysis is carried out on a BioResolve SCX mAb Column using certified
IonHance CX-MS pH Concentrates on the BioAccord LC-MS System.
The BioAccord
(Figure 1) is a
user-accessible
system comprised
of the ACQUITY
UPLC I-Class PLUS
System, TUV detector,
and ACQUITY
RDa Detector,
controlled with UNIFI,
a compliance-
ready software.
Online IEX-MS of mAb Charge Variants Using a BioResolve SCX mAb
Column, IonHance CX-MS pH Concentrates, and BioAccord System
Samantha Ippoliti, Andrew Schmudlach, Matthew A. Lauber, and Ying Qing Yu
Waters Corporation, Milford, MA, USA
Figure 1. BioResolve SCX mAb Column and BioAccord System
(ACQUITY UPLC I-Class with TUV System and RDa Detector,
controlled by UNIFI Software for acquisition and data processing).
[ APPLICATION NOTE ]
2
Online IEX-MS of mAb Charge Variants Using a BioResolve SCX mAb Column, IonHance CX-MS pH Concentrates, and BioAccord System
The I-Class provides robust chromatographic separation, and the RDa with SmartMS™ Technology delivers accurate mass information
with a simplified user experience.
This application note will show the broad applicability of the method on the BioAccord LC-MS System, as well as its value for
identifying charge variants formed upon forced degradation of mAbs. The ability to directly couple IEX separations to MS reduces
the dependency on fractionation for simple and peak identification.
EXPERIMENTAL
Sample preparation
Forced degradation of trastuzumab
A sample of trastuzumab (50 µL @ 20 mg/mL) was buffer exchanged into 100 mM sodium phosphate, pH 8.0 using BioRad Micro
Bio-Spin® chromatography columns (#732-6221), according to manufacturer protocol. The buffer exchanged trastuzumab sample
was further diluted to 2 mg/mL in 100 mM sodium phosphate, pH 8.0, and then split into two aliquots. One aliquot was frozen
at -80 °C until analysis, and the other was incubated at 25 °C for one week.
IdeS digestion of mAb samples
A 50 µg quantity of each antibody sample (NIST mAb, rituximab, infliximab, and trastuzumab [T0 and one week stressed]) was
digested by incubating at 37 °C for 30 minutes with 50 units of FabRICATOR® enzyme (Genovis, A0-FR1-008) in 25 mM NaCl,
25 mM Tris, 1 mM EDTA, pH 8.0 (with a final sample concentration of 1 mg/mL). A 1 mg/mL aliquot of each nonreduced antibody
sample was also prepared for analysis, and 10 µg of each sample was then injected for IEX-MS analysis.
LC conditions
LC system:
ACQUITY UPLC I-Class PLUS
Detectors:
ACQUITY TUV Detector,
ACQUITY RDa MS Detector
LC column:
BioResolve SCX mAb, 3 µm, 2.1 × 50 mm
with mAb Charge Variant Standard
(p/n: 176004342)
Column temp.:
30 °C
Sample vial:
12 × 32 mm Glass Vial, Total Recovery
(p/n: 600000750CV)
Mobile phase A:
10-fold dilution of IonHance CX-MS
pH Concentrate A (p/n: 186009280)
(50 mM ammonium acetate, pH 5.0,
2% acetonitrile)
Mobile phase B:
10-fold dilution of IonHance CX-MS
pH Concentrate B (p/n: 186009281)
(160 mM ammonium acetate, pH 8.5,
2% acetonitrile)
Gradient table (Intact mAb):
Time
Flow rate
(min) (min)*
%A
%B
Initial
0.100
60.0 40.0
1.00
0.100
60.0 40.0
21.00
0.100
2.0 98.0
22.00
0.100
2.0 98.0
23.00
0.100
60.0 40.0
30.00
0.100
60.0 40.0
Gradient table (IdeS Digest):
Time
Flow rate
(min) (min)*
%A
%B
Initial
0.100
98.0 2.0
1.00
0.100 98.0 2.0
21.00
0.100
2.0 98.0
22.00
0.100
2.0 98.0
23.00
0.100
98.0 2.0
30.00
0.100
98.0 2.0
*It is recommended to use a low dispersion ACQUITY UPLC I-Class
instrument for performing this type of gradient with a 0.1 mL/min flow rate.
If another instrument is to be used, it might be of benefit to gradient fidelity
to scale the method with an increase in flow rate to at least 0.15 mL/min.
[ APPLICATION NOTE ]
3
Online IEX-MS of mAb Charge Variants Using a BioResolve SCX mAb Column, IonHance CX-MS pH Concentrates, and BioAccord System
[ APPLICATION NOTE ]
RESULTS AND DISCUSSION
Until recently the investigation of charge variants required tedious fraction
collection and buffer exchange or a complex 2D-LC instrument setup to
acquire mass spectrometry data. Herein, a mobile-phase system based
on IonHance CX-MS pH Concentrates has been devised. With these
concentrates, optimized ammonium-based mobile phases can be quickly
prepared for MS-compatible IEX separations. The certified IonHance
CX-MS pH Concentrates are prepared as 10x strength buffers packaged in
one-liter trace metal certified low-density polyethylene bottles to ensure
uncompromised MS quality. Concentrate A is formulated to yield a pH 5.0
mobile phase, and Concentrate B is formulated to generate a higher ionic
strength pH 8.5 mobile phase.
The resulting mobile-phase system, BioResolve SCX mAb Column, and
BioAccord System make for a compelling new analytical approach for
the biopharmaceutical industry, where charge variant analysis is widely
employed for drug characterization and stability studies. Charge variant
profiles change over time or stress conditions, making them a critical quality
component. The ability to directly investigate new or increasing peaks in
the IEX charge profile with MS-compatible mobile phases saves time and
will reduce the necessity to send samples to specialized characterization
labs. In addition, it eliminates potential artificial degradation due to sample
manipulation during fraction collection.
This method was first established with NIST mAb, rituximab, and infliximab,
as shown in Figure 2, respectively. The ToF settings were tuned for the
optimal ionization of intact mAbs and subunits in native conditions. Source
parameters were based on a balance of MS-signal intensity and mass
resolution along with consideration of what conditions best preserve the
native state of the antibody and subunits. For NIST mAb and infliximab, the
prominent charge variants are related to the presence of C-terminal lysine.
With this method, the C-terminal lysine variants are well resolved via IEX
chromatography, and then their identity could be readily confirmed with MS
detection. Accordingly, this IEX method can be applied to a variety of mAb
samples without the need for extensive, individualized gradient optimization.
A 10 µg mass load on a 2.1 mm I.D. column was sufficient for detecting
variants down to at least a 1% relative abundance. In addition, the technique
produces high quality mass spectra with minimal interference caused by
formation of salt adducts.
ACQUITY RDa Detector settings
Mass range:
m/z 400–7000
Mode: ESI+
Cone voltage:
150 V
Desolvation temp.:
350 °C
Capillary voltage:
1.5 kV
Lock mass:
Leu-enkephalin at
50 fmol/µL in 50/50
water/acetonitrile
with 0.1% formic acid
Informatics:
UNIFI Scientific
Information System
[ APPLICATION NOTE ]
4
Online IEX-MS of mAb Charge Variants Using a BioResolve SCX mAb Column, IonHance CX-MS pH Concentrates, and BioAccord System
A
B
C
*
*
*
D
E
F
Figure 2. IEX-MS of intact mAbs. Left panel shows UV trace at 280 nm for (A) NIST mAb, (B) rituximab, and (C) infliximab.
Right panel (D, E, and F) displays combined native MS spectra for main peaks (denoted with “*”) for NIST mAb, rituximab,
and infliximab, respectively.
Figure 3. UV (280 nm) chromatogram overlay of T0 and one week
stressed trastuzumab after IdeS digestion. Representative integrations
are displayed for acidic and basic variants.
Figure 4. UV (280 nm) chromatogram overlay of T0 and one week stressed
trastuzumab at the intact level of analysis. Representative peak integrations
are displayed for acidic and basic variants.
As such, this IEX-MS method example is well suited to drug stability monitoring. To showcase this, we chose to investigate the forced
degradation study of trastuzumab, a mAb which is well known to easily deamidate under elevated pH and temperature. Figure 3
shows the IdeS digest of unstressed (T0) versus stressed trastuzumab. With the stressed condition, there is an increase of 14.1% in
acidic variants of the Fab region (Peaks C and D), and 4.5% of the Fc region (Peak A). The increase in acidic variants for IdeS digests
corresponds well with the increase in acidic variants observed in nonreduced analysis (18.7%), as shown in Figure 4.
(Fc/2)2
Acidic Main Basic
T0
7.1%
84.4% 8.5%
1wk25C
11.6% 81.2% 7.2%
+4.5%
-3.2% -1.3%
(Fab)2
Acidic
Main
Basic
T0
20.9%
70.3% 8.9%
1wk25C
34.9% 56.4% 8.7%
+14.1%
-13.8% -0.2%
A
B
C
D
E
F
T0
1 week @ 25 °C
UV @ 280nm
(Fc/2)2 Acidic
(Fc/2)2 Main
(Fc/2)2 Basic
(Fab)2 Acidic
(Fab)2 Main
(Fab)2 Basic
A
B
C
D
T0
Acidic
Main
Basic
1 week @ 25 °C
Acidic
Main
Basic
T0
14.0%
74.7% 11.3%
1wk25C
32.7% 58.3% 9.0%
+18.7%
-16.4% -2.3%
[ APPLICATION NOTE ]
5
Online IEX-MS of mAb Charge Variants Using a BioResolve SCX mAb Column, IonHance CX-MS pH Concentrates, and BioAccord System
Figures 5 and 6 display the online MS data
(combined spectra) collected for each of the
charge-variant species in IdeS digested and
non-reduced trastuzumab samples, respectively.
The main peaks in the IdeS digested sample
correspond to (Fc/2)
2 and (Fab’)2 species, and
their mass spectra are shown in Figures 5B and
5E, respectively. Observed masses can be seen
within 20 ppm of theoretical masses. Acidic species
shown in Figure 5A are likely deamidated (Fc/2)
2
species as well as the N-glycan species containing
sialic acid. The acidic species represented by
the spectra of Figures 5C and 5D are likely the
well characterized (Fab’)
2 deamidation species,
and species represented by Figure 5F is likely
a conformational variant. In the non-reduced
sample, Figure 6C corresponds to the main
species (within 20 ppm of calculated mass of the
trastuzumab G0F/G0F glycoform). Figures 6A–B
and 6D likely correspond to deamidated species
or conformational variants, respectively.
One apparent challenge in this analysis is
the assessment of isobaric and near-isobaric
species such as deamidation or conformational
differences. These charge variants have little or
no mass difference in comparison to the full-size
antibody, which makes it difficult to confidently
assign these variants by intact mass. Peptide
level characterization is required to confirm
with high confidence, if these peaks are true
isobaric or deamidated species. In the case of
trastuzumab, the deamidation susceptibility is
well characterized and expected at the levels
observed here for acidic variants.4 In practice,
the direct IEX-MS method is useful to corroborate
hypotheses about near-isobaric variants as well
as to rule out other possible variants.
Furthermore, the use of a chromatographic
method has a distinct advantage over other
charge-based separations such as capillary
electrophoresis or isoelectric focusing, because
it lends itself more easily to fraction collection
and to performing other types of testing. The
variant peaks separated by this IEX method
can still be collected and analyzed via peptide
mapping experiments to confirm the location
of the modification, or they can be isolated for
drug potency assays.
C
D
E
F
A
B
C
D
E
F
A
B
Observed mass [m/z]
Mass [Da]
Mass [Da]
Mass [Da]
Mass [Da]
Mass [Da]
Mass [Da]
Figure 5. The panel on the left shows combined raw spectra for peaks A–F in Figure 3
(IdeS-digested trastuzumab); the panel on the right displays the corresponding MaxEnt1
deconvoluted spectra.
Figure 6. The panel on the left shows raw spectra for peaks A–D in Figure 4 (intact level of
trastuzumab); the panel on the right displays the corresponding MaxEnt1 deconvoluted spectra.
D
C
B
A
D
C
B
A
Observed mass [m/z]
Mass [Da]
Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A.
T: 1 508 478 2000
F: 1 508 872 1990
www.waters.com
[ APPLICATION NOTE ]
Waters, The Science of What’s Possible, BioAccord, ACQUITY, UPLC, BioResolve, IonHance, UNIFI, and SmartMS
are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.
©2019 Waters Corporation. Produced in the U.S.A. September 2019 720006672EN AG-PDF
CONCLUSIONS
A direct IEX-MS method with broad utility has been successfully
implemented and applied to a case study on the detection of mAb charge
variants. Platform methods can be easily developed using the BioResolve
SCX mAb Column, IonHance CX-MS pH Concentrates, and BioAccord
LC-MS System which provide robust and reproducible separations along
with high quality mass spectra in the elucidation of various mAb-charge
variant species. The resulting workflow can increase efficiency for charge-
variant characterization and monitoring, as it circumvents the need for
fraction collection where MS-based peak assignment is sufficiently
straightforward. The simplified buffer preparation, robust chromatographic
separation, and clean MS spectra bring higher throughput and faster
decision making in the development of biopharmaceuticals.
References
1. Schmid, I.; et al. Assessment of Susceptible
Chemical Modification Sites of Trastuzumab
and Endogenous Human Immunoglobulins at
Physiological Conditions. Communications Biology
2018, 1 (28).
2. Diepold, K.; et al. Simultaneous Assessment of Asp
Isomerization and Asn Deamidation in Recombinant
Antibodies by LC-MS Following Incubation at
Elevated Temperatures. PLoS ONE
2012, 7 (1):
e30295.
3. Espinoza-de la Garza, C.; et al. Capillary
Electrophoresis Separation of Monoclonal Antibody
Isoforms Using a Neutral Capillary. J. Vis. Exp.
2017,
(119): 55082.
4. Giorgetti, J.; et al. Intact Monoclonal Antibodies
Separation and Analysis by Sheathless Capillary
Electrophoresis-Mass Spectrometry. European
Journal of Mass Spectrometry
2018, 25, 324–332.
5. Piešt’anský, J.; et al. Two-Dimensional Capillary
Electrophoresis with On-Line Sample Preparation
and Cyclodextrin Separation Environment for
Direct Determination of Serotonin in Human Urine.
Molecules
2017, 22 (10), 1668.
6. Shaeper, J.; et al. Parameters Affecting
Reproducibility in Capillary Electrophoresis.
Electrophoresis
2000, 21 (7), 1421–1429.
7. Yan, Y.; et al. Ultrasensitive Characterization
of Charge Heterogeneity of Therapeutic
Monoclonal Antibodies Using Strong Cation
Exchange Chromatography Coupled to Native
Mass Spectrometry. Anal. Chem.
2018, 90 (21),
13013–13020.
8. Leblanc, Y.; et al. Charge Variants Characterization
of a Monoclonal Antibody by Ion Exchange
Chromatography Coupled On-Line to Native Mass
Spectrometry: Case Study After a Long-Term
Storage at +5 °C. J. Chromatogr. B: Anal. Technol.
Biomed. Life Sci.
2017, 1048, 130–139.
