Care and use manual – Waters UPLC Intact Mass Analysis Application Kit User Manual
Page 7
Figure 8. Total ion chromatograms (TICs) from Waters ACQUITY UPLC-LCT
Premier ESI-TOF MS analyses of an intact IgG1, and pre and post blank runs.
Column temperature was set to 80 °C for these analyses.
Figure 9. Combined ESI-Tof (ACQUITY UPLC-LCT Premier ESI-Tof MS) mass
spectra of an intact IgG1 demonstrating regeneration to pre-injection
conditions.
b. Reduced Antibody Analysis
A ten minutes LC-ESI-MS method was used to resolve and
characterize IgG heavy and light chain subunits. For efficient
sample desalting, a system controlled post-column valve was used
for waste diversion of sample buffers and salts, prior to initiating
the analysis gradient. Additional sawtooth gradient cycles were
applied following the analysis gradient to regenerate the column
back to pre-injection conditions (Table 5). To minimize run cycle
time, and maximize system performance, higher flow rates
(0.5 ml/min) were applied for column regeneration. The 10 min
LC-MS run largely resolved the earlier eluting light chain from the
later eluting glycosylated heavy chains (Figure 10).
Table 5. Gradient profile used for reduced IgG1 analysis.
Figure 10. Total ion chromatograms (TICs) from LC-MS analyses of a reduced
IgG1, and pre and post blank runs. Combined ESI-TOF mass spectra of light and
heavy chains are shown in inset. Column temperature was set to 80 °C for
these analyses.
MASS Spectrum
Light Chain
MASs Spectrum
Heavy Chain
Total Ion
Chromatogram
(1:3 µg load)
Pre and Post-run
Blank
TIME
Pre-run Blank
0.5 µg IgG1
Post-run Blank
m/z
Max: 2 Counts
754 Counts
2 Counts
Pre-run Blank
0.5 µg IgG1
Post-run Blank
TIME
Time (min)
%B
Flow (ml/min) Curve
0.00
5
0.2
Initial
0.50
5
0.2
6
0.51
10
0.2
6
7.61
50
0.2
6
8.0
90
0.5
6
8.1
5
0.5
6
8.6
90
0.5
6
8.7
5
0.5
6
9.2
90
0.5
6
9.3
5
0.5
6
9.8
5
0.5
6
A= 0.1 % Formic acid (Water)
B= 0.1 % Formic acid (ACN) Column temp.: 80 °C
Load/Wash-
Divert Flow
Gradient
Column
Washing and
Regeneration
7
[ CARE AND USE MANUAL ]
UPLC Intact MASS Analysis Application Kit