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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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