Waters MassPREP Online Desalting Cartridge - Manual (Page 3)

IV. SUGGEST ED MET HOD FOR MASS ANALYSIS
USING MASSPREP ON-LINE DESALTING CARTRIDGE
(Considerations to Minimize Protein Carryover
Between Injections):
HPLC injector design, composition of injector wash solution, and
number of injector wash cycles between injections are some of the
factors that can effectively minimize protein carryover between
sample analyses. Waters MassPREP On-line Desalting Cartridges,
when used with an appropriately performing HPLC System and
injector wash protocol, can be used for the sequential analyses of
different samples due to the lack of protein carryover from prior
injections. Figure 3 shows the mass analyses results obtained
from buffer blank and intact protein injections [bovine serum
albumin (BSA) and IgG monoclonal antibody (mAb)] processed
through the MassPREP On-line Desalting Cartridge using the
Waters instrumentation and separation method detailed below:
a. Instrumentation
HPLC System: Alliance ® 2796 Separations Module
Needle Wash
Solution Composition:
H
2
0 (35%) / Isopropanol (5%)
Acetonitrile (60%)
Number of Needle Washes: Single
Needle Wash Time: 15 sec needle exterior/3 sec inject port
Mass Spectrometer: Q-tof
Ionization mode: ES +
Capillary voltage: 3300
Cone voltages: 30 for BSA mass analysis 35 for
mAb mass analysis
Source temp: 150 °C
Desolvation temp: 350 °C
Gas flow: 500 L/Hr
Cone gas: 50 L/Hr
b. Separation method:
Eluent A: H
2
0 with 0.1% Formic Acid
Eluent B: Acetonitrile with 0.1% Formic Acid
Flow: 0.4 mL/minute
Initial Column Conditioning:
Prior to use, the MassPREP On-line Desalting Cartridge reversed
phase packing material must be “wet” with organic solvent prior
to equilibration with 95% Eluent A/5% Eluent B. This can be
accomplished by running a “blank injection” (i.e., buffer injection
followed by running the separations gradient) or by washing the
cartridge with a minimum of five column volumes of 50% Eluent
B followed by a ten cartridge volume equilibration at 95% Eluent
A/5% Eluent B prior to sample injection.
Sample Loading/Salt Elution:
Load sample onto cartridge while delivering 95% Eluent A/5%
Eluent B. The protein sample will bind to the cartridge while the
contaminating salt passes through unretained to waste.
Sample Elution to Mass Spec:
At approximatly. 0.5 min, begin increase Eluent B concentration
from 5 to 80% in 1.5 min. Hold at 80% B for an additional 1.0 min
to allow protein to completely elute from cartridge into MS system.
Column re-equilibration:
At approximately 3.0 min, re-equilibration cartridge with ten
cartridge volumes of 95% Eluent A/5% Eluent B prior to next
sample injection.
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MassPREP On-Line Desalting Cartridge