1. * CORRESPONDING AUTHOR Bioanalysis of Exenatide: Intact Versus Signature Peptide Approach to Reach Optimal Sensitivity in Large Molecule Quantification by LC-MS Jean-Nicholas Mess, Daniel Villeneuve and Fabio Garofolo * Algorithme Pharma Inc., Laval (Montréal), QC, Canada INTRODUCTION CONCLUSION LC-MS is an emerging technique for the quantification of large molecules such as therapeutic peptides, proteins and biomarkers. However, due to the limited mass range of triple quadrupole mass spectrometers, these analytes are typically monitored as multicharged species. This dispersion of MS signal combined with poor fragmentation (CID) efficiency may affect the achievable LOQs. Another approach is to generate smaller signature peptides through tryptic digestion. These tryptic peptides will generally show less charge state distribution and clearer CID pattern than the intact analyte. However, the efficiency of the digestion process could be poor and compromise its sensitivity. In this study, both approaches will be compared in order to reach optimal sensitivity for the quantification of exenatide in human plasma. METHODS OVERVIEW PurposePurpose –Direct comparison of two different quantification approaches: intact versus signature peptide to reach optimal sensitivity for the quantification of exenatide in human plasma. MethodMethod –Sensitivity comparison was performed using reference standard material. –Samples were extracted by SPE, digested with trypsin and analysed by LC-MS/MS on an AB SCIEX QTRAP®5500 operated in ESI+ ResultsResults –The detection of the signature peptide was 5 times more sensitive than the intact exenatide. –The digestion efficiency was evaluated to be >80% –Using the digestion approach, an LLOQ of 10 pg/mL was achieved with an assay showing good precision and accuracy. SAMPLE EXTRACTION Range of 10 – 2000 pg/mL in human plasma 500 µL of sample extracted by solid phase extraction using Oasis ion exchange SPE. Following elution and drying step, the samples were reconstituted with trypsin (Sigma T1426) and incubated for 3 hours at 37°CCHROMATOGRAPHY Agilent Technologies Series 1100 pumps and autosampler Ascentis Express C18, 50x2.1mm, 2.7µm Gradient elution of 0.1% HCOOH and ACN 3.5 minutes run timeDETECTION AB SCIEX QTRAP®5500 MRM mode ESI(+) The [M+5H] 5+ (m/z 838.3 → 396.2) was monitored for exenatide and the [M+2H] 2+ (m/z 475.1 → 688.4) was monitored for the signature peptide. Table 1 Exenatide amino acids sequence analysis showed 3 theoretical trypsin cleavage sites which would result in the generation of 4 tryptic peptides. However, experimental digestion data performed in solution showed the presence of only 3 tryptic peptides. One of the tryptic peptide (LFIEWLK) had good characteristic for quantification (retention on C18 column and MS sensitivity) (Table 1). This peptide mainly ionized to the +2 charge state (m/z 474.8) and clearly fragmented to one singly charged product ion (m/z 688.4). Table 2: Summary of the Evaluations Performed Figure 4: Representative Chromatograms of Extracted and Digested Exenatide in Human Plasma RESULTS A) No Trypsin Added B) Following Digestion (3 hrs at 37°C) Figure 3 The extraction efficiency of exenatide (SPE process) was on average 60.9% and the digestion efficiency was 80.4%. No intact exenatide was found in the extracted samples following tryptic digestion (Figure 3). Overall, the entire extraction efficiency process (SPE + digestion) was found to be 49.0%. Figure 4 The lower limit of quantification (LLOQ) achievable using the tryptic digestion approach was 10 pg/mL (Figure 4), which is significantly lower than the reachable LLOQ for the intact exenatide. A) Extracted Blank B) Extracted LLOQ (10.0 pg/mL) C) Extracted ULOQ (2000.0 pg/mL) This study demonstrates that the tryptic digestion approach is suitable for the quantification of exenatide in human plasma by LC-MS/MS. The digestion step was efficient (within 3 hours) and reproducible. The tryptic peptide generated allowed to decrease the LLOQ of the exenatide assay. The LLOQ achieved by this LC-MS/MS assay is comparable, if not lower, to the commercial LBA assay kits. On-going work is currently being performed on the novel triple quadrupole QTRAP®6500 to decrease the LLOQ to the low pg/mL level. Figure 1: Structure of Exenatide and Signature Peptide PeptideCharge State Monoisotopic (m/z) t R (min) Intensity (cps) HGEGTFTSDLSK+11278.5961Outside calibration +2639.80170.638.84e5 +3426.87020.63 7.73e5 QMEEEAVR+1991.4513 Not Detected +2496.2293 +3331.1553 LFIEWLK+1948.5553Not Detected +2474.78132.335.37e6 +3316.85662.335.74e4 NGGPSSGAPPPS-NH 2 +11023.4854Not Detected +2512.24630.347.30e5 +3341.83330.341.56.e4 Table 1: Exenatide Tryptic Digestion Figure 2 When comparing the synthetic reference standard material for exenatide and its tryptic peptide prepared at an equimolar concentration (Figure 2), the detection of exenatide tryptic peptide was 5 times more sensitive than the intact exenatide. Intact exenatide S/N = 84.2 Signature peptide S/N = 431.5 Intensity (cps) Figure 2: Sensitivity comparison of Intact Exenatide and Exenatide Signature Peptide Prepared at Equimolar Concentration (1 nM) Figure 3: Tryptic Digestion Efficiency of Exenatide to its Signature Peptide in Human Plasma SPE Extracts Signature Peptide Exenatide Exenatide Intensity (cps) Figure 5 Table 2 The calibration curve was linear (weighted 1/x 2 ) from 10.0 to 2000.0 pg/mL with a coefficient of correlation of 0.9951 (Figure 5). Summary of the preliminary data obtained during method development is shown in Table 2. Intensity (cps) Concentration (pg/mL) Peak Area Ratio Regression type: Linear 1/x 2 Coefficient Correlation: r = 0.9951 Figure 5: Calibration Curve for Exenatide EvaluationResults Inter Batch Precision (%CV) and Accuracy (%Bias)8.2% and 11.7% at LLOQ QC 7.1% and 2.2% at Low QC 3.7% and 1.7% at Mid QC 4.6% and 3.0% at High QC Extraction Process (SPE) Yield64.7% at Low QC 57.1% at High QC Digestion Process Yield75.3% at Low QC 85.5% at High QC Total Process Yield48.7% at Low QC 48.8% at High QC Matrix Effect Acceptable for 9/10 donors of human plasma including lipemic samples. Hemolysed samples under investigation. SelectivityNo significant interference at analyte and IS retention times for 10 donors including lipemic and hemolysed samples Dilution Integrityup to 4 000 pg/mL, diluted 5-fold Processed Stability45.8 hrs at 4°C Bench-Top Stability (K 2 EDTA + Inhibitor)20.0 hrs on ice/water bath 2.2 hrs at room temperature Long-Term Storage Stability (K 2 EDTA + Inhibitor)On going Freeze-Thaw Stability (K 2 EDTA + Inhibitor)3 Freeze Thaw Cycles Stability in Whole Blood (K 2 EDTA)2.0 hr on ice/water Stock Solution Stability 100 µg/mL (Days)48 days at 4°C in aq.solution