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Table 1. Quantification Curve for Standard Ado-Trastuzumab Emtansine using Traditional Flow and Microflow LC-MS/MS. MultiQuant analysis based on peptide IYPT- NGYTR resulted in an accurate quantification with single digit CV% and r value of 0.995. Microflow LC-MS/MS provides up to 5x increase in sensitivity with wider linear
dynamic range as compared to Traditional LC-MS/MS.
provides a uniquely selective and sensitive method for quantification. For the signature peptides chosen here, DiscoveryQuant™ software was used to aid in the selection and optimisation of the best product ions for each peptide precursor. The software will automatically ramp MS parameters in a series of experiments to find the best MS conditions and fragmentation energies for each MRM transition, greatly expediting the development of the final assay.
Figure 5. Quantification Curve for Standard Ado-Trastuzumab Emtansine using Signature Peptide IYPT- NGYTR. MultiQuant quantification curve using peptide IYPTNGYTR resulted in an accurate quantification
with single digit CV% and r value of 0.995 using peak area and 1/x^2 weighting.
included in the kit and provides a universal means to capture human mAbs. It will capture all ADC species including those without payload attached and provides a total antibody measurement. After capture and elution from the beads, samples are reduced, alkylated, and digested with a Trypsin/Lys C enzyme combination using reagents and buffers included in the BioBA kit which includes a mass spec compatible anionic surfactant (Figure 3). Because the samples also contain the SILuMab internal standard (IS), this protein is also digested. The digestion efficiency of the IS does not need to be equivalent to the ADC as long as it is reproducible.
Once the protein has been digested, one or more peptides are used as surrogates for protein quantification. The ado- trastuzumab emtansine conjugation is through lysine groups but in general, lysine containing peptides should be avoided as their cleavage may not be reproducible. The signature peptides IYPTNGYTR and
FTISADTSK from the CDR region and the conserved Fc peptide DTLMISR are typically chosen for quantification of trastuzumab. However, since the peptide FTISADTSK contains a lysine, the two signature peptides IYPTNGYTR and DTLMISR are preferred for ado-trastuzumab emtansine. Peptide IYPTNGYTR is a unique peptide to trastuzumab from the CDR region, peptide DTLMISR is a universally conserved human IgG peptide. The SILuMab internal standard (IS) yields a heavy labelled peptide for DTLMISR. This heavy labelled peptide was used as the IS for all signature peptides used for quantification as it is very close in retention time to the analyte peptides.
The most effective LC-MS/MS assays for quantification use a technique called multiple reaction monitoring, or MRM. In MRM, the precursor ion of a specific analyte is selected in the first mass analyser, fragmented in a collision cell, and then one or more of the resulting product ions are monitored in the second mass analyser. This
Figure 4 shows the extracted ion chromatograms (XICs) of the signature peptide FTISADTSK at the 5 ng/ml and 10 ug/ml level for both traditional flow and microflow LC. The S/N ratio was improved by 4 fold for this signature peptide as well as IYPTNGYTR using microflow LC.
Table 1 shows the comparison of the quantification statistics generated using MultiQuant Software for data acquired by traditional and microflow LC for the signature peptide IYPTNGYTR. Figure 5 shows the calibration curve for this same peptide using Microflow LC. The LLOQs for both methods were determined using the requirements of precision < 20% and accuracy between 80 and 120% at LLOQ, and at any higher concentration a precision < 15% and accuracy between 85% and 115%. LLOQ improved by a factor of 5 using the microflow LC trap-and-elute method using both signature peptides and the conserved Fc peptide DTLMISR. For the traditional LC method the limit of quantification LOQ was 5 ng/ml whereas an LOQ of 1 ng/ml was achieved by microflow LC. Both traditional flow and microflow LC methods showed good linearity with r > 0.99.
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