3
Thermo Scientific Poster Note
•
PN-64082-ASMS-EN-0614S
platform for a monoclonal
th high resolution Orbitrap mass
urement of mAb, its aggregates,
an be measured by SEC-MS
H eluent
ass spectrometer enables the
ssfully separates the HC and
ing denaturing eluent.
on the development of
bs produced from mammalian
and higher-order aggregates.
ed technique for the detection
logical drug products. It is
rol of mAb products.
ion as well as the glycan profile
on mass spectrometry. The most
Ab via reversed phase
r, the extreme low pH and
phy often denatures the mAb. In
ain cysteine linked drugs, the
ght chains of the ADC and
ring SEC-based desalting mass
he mAb in its native state. The
S and preserves intact protein
mass of the mAb fragments,
ed by reduction of interchain
apain digestion. Using
A, and 0.05% formic acid, SEC
separate Fab and Fc. It serves
high-resolution, accurate mass
/z
range up to 20,000 and
r signals. All these features
erb tool for accurate intact mass
of mAb glycosylation profile.
y (SEC) column designed for
ers, aggregates, and fragments.
nd compatibility with MS
ity of mAbPac SEC-1 with
les intact mass detection of
condition and fragments
denaturing conditions.
Separation of mAb2 dimer aggreg
column (2.1
㽢
150 mm) within 8
were successfully detected (Figur
aggregates show dimer peaks at
corresponding to the homo-dimer
(Figure 2e). The mass difference
derived from the monomer mass
aggregate peak at mass 296,949
mass 148,393 u and 148,554 u.
FIGURE 1: SEC-MS analysis of
20 mM NH
4
HCO
2
.
mAb was injec
the flow rate was set at 200 µL/mi
spectrum of mAb, (c) deconvolute
Liquid Chromatography
HPLC experiments were carried out using a Thermo Scientific™ Dionex™
UltiMate
™
3000 RSLCnano System equipped with: SRD-3400 Membrane Degasser, NCS-
3500RS dual-gradient pump and column compartment , and WPS-3000TPL Rapid
Separation Thermostatted Autosampler. SEC analysis was carried out in isocratic
mode. For the 4.0 mm ID column, flow rate was set at 200 µL/min. For the 2.1 mm ID
column, flow rate was set at 50 µL/min.
Reduction of mAb to heavy chain (HC) and light chain (LC) subunits
Reduction of inter-chain disulfides in a mAb (1 mg/mL) was achieved by incubation of
mAb with 20 mM DTT at 50
C for 30 min. The reduced sample was acidified with
formic acid to final concentration at 0.1%.
Papain digestion of mAb to generate Fab and Fc subunits
The digestion was carried out by incubating mAb (1 mg/mL) with papain (0.04 mg/ml)
in 100 mM Tris-HCl, pH 7.6, 4 mM EDTA and 5 mM Cysteine buffer at 37
C. After
4 hours, the digestion was stopped by addition of formic acid to final concentration at
0.1%.
Non-denaturing SEC mobile phase
20 mM ammonium formate (pH 6.3).
Denaturing SEC mobile phase
20% acetonitrile, 0.1% formic acid, and 0.05% trifluoroacetic acid acid (TFA).
MS Conditions
The Exactive Plus EMR mass spectrometer was used for this study. Intact mAb or
mAb fragments were analyzed by ESI-MS. HESI probe was used. See Table I for
details.
Data processing
Full MS spectra of intact mAbs, HC, LC, Fab, and Fc fragments were analyzed using
Thermo Scientific™ Protein
Deconvolution software (v 3.0) that utilizes the ReSpect
algorithm for molecular mass determination.
ased from Sigma
®
. Other reagents
antibodies mAb1 and mAb2 were
0
2
4
6
8
0
50
100
Relative Abundance
4000
4400
4800
5200
0
50
100
Relative Abundance
548
5287.83
5105.51
5310.31
4941.10
+2
+28
+29
G0
G0
Results
Analysis of mAb by non-denaturing SEC-MS
The analysis of mAbs by SEC is typically performed under non-denaturing conditions
at near-physiological pH range (6.8). The commonly used buffer is phosphate buffer
with 300 mM NaCl. However, the non-volatile nature of phosphate buffer and high salt
content makes this buffer non-compatible with online mass spectrometry detection.
Therefore, we explored using a volatile buffer such as 20 mM ammonium formate for
SEC separation and directly coupling the SEC column to the Exactive Plus EMR
instrument. Figure 1 shows the SEC-MS analysis of mAb1, with Figure 1a showing
the extracted ion chromatogram of
m/z
at 5483.08-5483.31 and Figure 1b showing the
charge envelope of +24 to +29 in the
m/z
range of 5100-6200. Normally under acidic
condition, the charge envelope of mAb is in the
m/z
range of 2000-4000. Since the
20 mM ammonium formate eluent has near neutral pH (at 6.3), the charge envelope of
mAb shifts to higher mass range. The detection of such high
m/z
charge envelope
(
m/z
above 6000) is made possible with the extended mass range of the Orbitrap
instrument. Figure 1c shows the deconvoluted mass spectra of the mAb, with a main
peak at mass 148,033 u and adjacent peaks at mass 148,198 u, and mass148,359 u,
corresponding to different glycoforms with 1 and 2 additional hexoses. The adjacent
peak at mass 148,163 u, is 130 u above the main peak, corresponding to a lysine
variant.
TABLE 1. MS conditions.
The Exactive Plus EMR Orbitrap mass spectrometer was
used for this study. The following parameters were employed for all measurements:
Source DC Offset: -25V; Injection Flatapole DC: -8V; Inter Flatapole Lens: -7V; Bent
Flatapole DC: -6V. Other ion transfer parameters in the tune file were set as default.
FIGURE 2: SEC-MS analysis of
denaturing condition using 20
SEC-1 2.1 x 150 mm column and
chromatogram of mAb monomer a
deconvoluted spectrum of mAb di
deconvoluted spectrum of mAb m
Instrument Conditions Non-denaturing, MAb Monomer Non-denaturing, MAb Dimer Denaturing
EMR mode
On
On
On
Mass range
m/z 400–20,000
m/z 2,000–15,000
m/z 400–6,000
Spray voltage
4.3 kV
4.3 kV
4.3 kV
Sheath gas
30 arb. units
15 arb. units
30 arb. units
Auxiliary gas
10 arb. units
3 arb. units
10 arb. units
Capillary temperature 275 °C
275 °C
275 °C
S-lens level
200
200
200
In-source CID
100 eV
100 eV
100 eV
HCD CE
10
100
n/a
Microscans
5
5
1
AGC target
1 × 10
6
3 × 10
6
1 × 10
6
Maximum IT
300 ms
200 ms
200 ms
Resolving power
35,000
17,500
17,500
Probe temperature
400 °C
100 °C
200 °C
(a)
(b)
(c)
+38
+37
+39
+40
G0+G1
2G0
2G1
