Care and use manual – Waters UPLC Intact Mass Analysis Application Kit User Manual
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VI. SUGGEST ED MET HOD FOR MASS ANALYSIS
USING UPLC INTACT MASS ANALYSIS
AP PLICATION KIT
Mass Spectrometry has become a powerful tool for therapeutic
proteins analysis. However, most therapeutic proteins are
stored in a matrix of biological buffers and non-volatile salts
and stabilizers. Thus, one of the most significant challenges
encountered during mass analysis of these proteins is processing
of the sample to remove these agents, which often form non-
covalent adducts that reduce MS response and further complicate
the resulting mass spectral data. The following offers two rapid,
sensitive and efficient desalting LC-MS methods that can be
utilized as a starting point with the UPLC Intact Mass Analysis
Application Kit for the characterization of an intact antibody and
its variants, and, constituent heavy and light chain structures.
a. Intact Antibody Analysis
A fast (4-min cycle time) and efficient LC-ESI-MS method was
used to profile multiple structural variants of an IgG. To minimize
cycle time, and maximize system performance, higher flow
rates (0.5 ml/min) were used for loading, desalting, and column
regeneration. A system controlled post-column valve was used
for waste diversion of sample buffers and salts. Additional
sawtooth (rapid) gradient cycles were applied to regenerate the
column to pre-injection conditions as part of each analysis (Table
4). Overlaid TICs (y-axis linked) for this experiment and the
associated summed mass spectra are shown in Figures 8 and 9,
respectively.
Table 4. Gradient profile used for intact IgG1 analysis.
Load/Wash-
Divert Flow
Gradient
Column
Washing and
Regeneration
Time (min)
%B
Flow (ml/min) Curve
0.00
5
0.5
Initial
0.50
5
0.5
6
0.51
5
0.2
6
2.00
90
0.2
6
2.10
5
0.5
6
2.70
90
0.5
6
2.80
5
0.5
6
3.40
90
0.5
6
3.50
5
0.5
6
4.00
5
0.5
6
A= 0.1 % Formic acid (Water)
B= 0.1 % Formic acid (ACN) Column temperature: 80 °C
MS
UPLC
Gradient
Column Heater
22.20”
8.5”
MS
Waste
UPLC
Gradient
SQD Divert Valve
8.5”
Waste
Position 1: Sample loading and salt divert to Waste
Position 2: Sample elution to MS
Figure 7. Fluidic configuration for LC-MS (UPLC-SQD) analysis. A post-column diversion valve located at the top right corner of the SQD
(typically used to direct calibrant for use in automatic tuning), was utilized to divert buffers and non-volatile salts to waste at the beginning
of the LC-MS analysis.
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UPLC Intact MASS Analysis Application Kit