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Characterization Quality Control Pharmacokinetics
Evaluating the Concentration of Antibody Drugs in Blood Nexera Mikros
High-Sensitivity LC/MS Bioanalysis of benefits
Trastuzumab by nSMOL Cell Line Optimization
click here • Micro-flowrate LC-MS system that offers both high sensitivity and high throughput.
• Easy one-step attachment of analytical columns and connection to the LC-MS
Operating Principle and Features Table 3 MRM Transitions of Quantified Peptides in Trastuzumab
Peptide MRM transition Purpose ionization interface.
High-performance liquid chromatograph mass spectrometer (LC-MS) P 14 R 512.1 > 292.3 (b3+) For quantitation (IS)
systems enable higher performance analysis by decreasing the flowrate 512.1 > 389.3 (b4+) For structural confirmation
in the LC unit and improving the ionization and ion uptake efficiency 512.1 > 660.4 (b6+) For structural confirmation Direct injection system: Small injection volumes make it especially well-suited for analyzing desalted or otherwise Culture
in the MS unit. The Shimadzu Nexera Mikros is a micro LC-MS system IYPTNGYTR 542.8 > 404.7 (y7++) For quantitation pretreated samples.
For structural confirmation
542.8 > 808.4 (y7+)
that reduces the LC flowrate to a micro level (approx. 1 to 10 μL/ 542.8 > 610.3 (y5+) For structural confirmation
min). That results in between several times to several tens of times Note: The peptide (signature peptide) was selected from trypsin peptide fragments that include
higher sensitivity than the previous semi-micro LC-MS system, while complementarity-determining regions (CDRs) that determine antibody specificity.
maintaining the same robustness and throughput.
Results
Measurement Method and Conditions
Measured concentrations of trastuzumab in blood plasma correlated
The ELISA ligand-binding assay method was the primary method closely with concentration settings, with an R value of 0.99 or higher
2
used to determine the concentration of antibody drugs in the blood. and excellent accuracy and precision. Furthermore, the Nexera Mikros Purification
This example describes a quantitative method that is based on using system was also used to confirm that the LLOQ value for trastuzumab
a high-sensitivity LC-MS system. Human blood plasma spiked with in blood plasma is 0.00763 μg/mL. Trap and elute system: System for increasing sensitivity of semi-micro systems without changing the injection volume or
a trastuzumab standard and blank blood plasma were analyzed as A comparison of MRM chromatograms from the Nexera Mikros system other parameters
samples. For all the antibody drugs, Fab-derived peptides were obtained (Fig. 1) and a typical semi-micro LC-MS system (Fig. 2) shows that the
using the nSMOL method, which allowed using the same protocol Nexera Mikros is able to detect with ample sensitivity concentrations
for all samples. These were analyzed to quantify the concentration of that the semi-micro LC-MS system was not able to detect.
trastuzumab in the blood plasma based on the analytical conditions
indicated in Tables 1 to 3.
Table 1 LC Analytical Conditions
[Analytical]: Characterization
Column: Shim-Pack MC C18
(50 mm × 0.175 mm I. D., X µm)
Mobile phase A: 0.1 % Formic acid in water
Mobile phase B: 0.1 % Formic acid in Acetonitrile
Gradient: 5 % (0 – 0.5 min) →
(B. Conc. ) → 25 % (0.5 – 4.5 min) →
→ 95 % (4.51 – 5.0 min) → Ionization promotion system: System for increasing negative mode sensitivity by adding an LC-20AD nano unit to each
→ 5 % (5.01 – 11.0 min) of the two systems above
Flowrate: 4.0 µL/min
Column Temp.: 50 °C
[Trap]
Trap column: L-column 2 ODS Micro Quality Control
(5 mm × 0.3 mm I.D., X µm)
Mobile phase A: 0.05 % Trifluoroacetic acid in water
Mobile phase B: 0.1 % Formic acid in Acetonitrile
Column Temp.: 50 °C
Injection Volume: 10 µL
Table 2 MS Analytical Conditions Fig. 1 MRM Chromatograms (Nexera Mikros)
Ionization: ESI Positive
DL Temp.: 250 °C
Heat Block Temp.: 400 °C
ESI Temp.: 100 °C Pharmacokinetics
Nebulizer Gas: 2 L/min
Drying Gas: OFF
Heating Gas: 3 L/min
Specifications
Conclusion Solvent delivery unit LC-Mikros Flowrate range: 1 to 500 μL/min
Using the nSMOL method in combination with the Nexera Mikros System pressure capacity 80 MPa
system enables high-sensitivity quantitation of antibody drugs in blood Fig. 2 MRM Chromatograms (Semi-Micro LC-MS) Autosampler SIL-40C XR Injection volume range: 0.1 to 50 µL Others
without sacrificing throughput.
Application Examples Ionization unit Micro-ESI 8060 or Micro-ESI 9030
Column oven CTO-Mikros
• Analyzing lipid mediators with high sensitivity
(UF-Link enables connecting/disconnecting columns easily with zero dead volume.)
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