Waters Solutions for DMPK and Biomarker Analysis Waters User Meeting ASMS 2011 Denver, Colorado Stephen McDonald 2011 Waters Corporation 1
Drug Development Process Pre-clinical testing R&D - 18 months Candidate Selection & Confirmation Clinical R&D - 5 years Phase 1 Phase 2 Phase 3 Submit IND Submit NDA 2011 Waters Corporation 2
Instrument Needs High throughput Medium/High sensitivity assays Simple validation required Moderate throughput Highest sensitivity assay Full validation required High throughput Medium/High sensitivity assay Full validation required Pre-clinical testing R&D - 18 months Candidate Selection & Confirmation Clinical R&D - 5 years Phase 1 Phase 2 Phase 3 Submit IND Submit NDA QTof Quad 2011 Waters Corporation 3
Rationalizing Instrument Deployment Question? Precisely how much do I have? Study PK/PD ADME Instrument Triple Quads Question? What is it? Study Metabolite ID Biomarker Analysis Instrument Q-Tof Benefits MRM, High Sensitivity, High Selectivity Benefits Full scan acquisition, accurate mass 2011 Waters Corporation 4
Rationalizing Instrument Deployment Question? What is it and how much of it is there? Studies Met ID for MIST Identifying metabolites in a PK study Biomarker studies where performing in vivo analysis Exploit mutual benefits but only where it makes sense 2011 Waters Corporation 5
Drug Metabolism PK/PD & ADME Toxicity (non-glp) 2011 Waters Corporation 6
Ideal Scenario for Discovery DMPK All peaks detected No Method Development Identify all metabolites Fast analysis No need for expert users 100 Linear Response 80 Quantify with Response 60 40 20-0 ng/ml -0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 2011 Waters Corporation 7
The tools that power the discovery DMPK solution 2011 Waters Corporation 8
Nefazodone MS E Analysis 2011 Waters Corporation 9
Using Chemical Intelligence Cleavages unique to molecule itself Potential Biotransformations +O +glucuronide +H 2 O +sulfate +O 2 +glutathione +.. +.. +.. +.. H60 10 µm, S+ MSe 100 0% Data (Fragmentation Spectra) x10 290.1516 124.0517 152.0834 156.0786 178.0996 188.1090 189.1125 209.0862 218.0939 219.0956 237.1141 246.1251 262.1190 272.1407 m/z 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500 486.2277 291.1547 488.2251 Need to bring all this knowledge together in a meaningful way and have a clear way of visualizing this to the user 2011 Waters Corporation 10
Applying Chemical Intelligence 2011 Waters Corporation 11
Where Is My Hydroxylation? 2011 Waters Corporation 12
Automated Localization of Biotransformation 2011 Waters Corporation 13
Accurate Mass Quantification of Nefazodone @ 50pg/mL in Plasma 0.2 260_19_6_004 100 6.52 7.15 1: TOF MS ES+ 260.16 953 0.48 0.45 % 0.43 0.98 1.99 2.50 2.68 3.69 11.42 11.39 11.48 11.52 0.27 0.92 8.85 11.60 11.71 0.65 1.49 1.92 1.24 2.35 2.88 2.95 3.39 3.98 4.35 4.69 4.78 6.23 6.15 5.37 5.64 5.83 6.83 7.53 7.80 8.14 8.33 8.71 9.43 9.18 9.73 10.05 10.23 10.85 11.27 11.78 11.95 0 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 Time 2011 Waters Corporation 14
Accurate Mass To Detect Peak in Complex matrix 100 3.69 1Da Window 260.16 Nominal Mass % 3.95 3.49 4.11 4.21 0 3.50 3.60 3.70 3.80 3.90 4.00 4.10 4.20 4.30 4.40 4.50 100 3.94 260.16 0.05Da 50mDa Window Accurate Mass % 0 Time 3.50 3.60 3.70 3.80 3.90 4.00 4.10 4.20 4.30 4.40 4.50 2011 Waters Corporation 15
Linear Response Over 4 Orders of Magnitude Sensitivity in the 50pg/mL range Fast data acquisition for narrow UPLC peaks Linear response > 4 orders of magnitide 100 80 Response 60 40 20-0 ng/ml -0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 2011 Waters Corporation 16
Fragment Ions: MS E Elevated Energy Data 100 260.1645 1.07e 260.1651 116.1071 116.1071 183.0810 % 183.0803 157.0647 72.0814 74.0608 98.0968 157.0647 184.0743 264.2688 104.1074 165.0702 117.1104 265.2719 153.0702 168.0571 218.1175 56.0506 301.1508 86.0968 118.1137 142.9403 185.0771 59.0698 182.0732 207.9989 242.1552 258.1132 284.1238 297.2391 0 m/z 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 2011 Waters Corporation 17
Calibration Lines Using Fragment Ion and Precursor Ion LLOQ = 500pg/mL m/z = 116.107 (10mDa) Compound name: propranolol high energy Correlation coefficient: r = 0.999660, r^2 = 0.999319 Calibration curve: 0.0285934 * x + 12.0348 Response type: External Std, Area Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None 2500 2000 Response 1500 1000 500-0 pg/ml -0 20000 40000 60000 80000 100000 m/z = 183.081 (10mDa) LLOQ = 500pg/mL Compound name: propranolol high energy Correlation coefficient: r = 0.997492, r^2 = 0.994991 Calibration curve: 0.0204225 * x + -0.136933 Response type: External Std, Area Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None 2000 1800 1600 1400 Response 1200 1000 800 600 400 200-0 pg/ml -0 20000 40000 60000 80000 100000 2011 Waters Corporation 18
Accessible Quantitative Analysis 2011 Waters Corporation 19
ADME Profiling In Drug Discovery Sample Formatting Perform ADME (stability, solubility etc) Method Development Generate LC/MS/MS methods Run Samples Run standards and samples Quantify Quantify samples Data Generation Calculate data - solubility, stability, protein binding etc. Report Results Rank samples and report to project team 2011 Waters Corporation 20
ADME with Xevo TQ(S) ACQUITY UPLC Fast, robust method development to high throughput screening Xevo TQ(S) MRM sensitivity Software Drive efficiency and Productivity 2011 Waters Corporation 21
Fast p450 Screening by UPLC/MS/MS & Xevo TQ-MS 1. acetaminophen 2. 1-hydroxybufuralol 3. 4-hydroxymephytoin 4. 7-hydroxy Coumarin 5. 4-hydroxytolbutamide 6. 1-hydroxymidazolam 1 2 3 4 5 6 P450 inhibition and induction testing allows drug drug interactions and potential metabolic issues to be identified and eliminated 28 sec 7.8 sec Time 60 min Control UPLC and Xevo TQ facilitates rapid analysis with resolution between all the p450 probe substrates Time 60 min 0.1 µm Time 60 min 1.0 µm Time 60 min 10 µm 2011 Waters Corporation 22
High Throughput Quantitative Analysis Xevo TQ and QuanOptimize Sample Plate Preparation (ROBOTICS) Optimise MRM transition Cone Voltage and Collision Energy Build MRM method (inc internal standard) Generate Quantification Method Acquire Quantification Data Fully Automated by QuanOptimize Data Process and Generate Report 2011 Waters Corporation 23
OpenQuan - Open Access Optimization and Quantitation Login User name, job ID, e-mail Finish Results by e-mail or print Next user Select Quan Method Set by admin Position Samples Indicted by Login screen Submit Optimisation List Import or cut-andpaste Submit Acquisition List Import or cut-andpaste 2011 Waters Corporation 24
Handling of Multiple Assay Results with ProfileLynx DATA PROCESSING AND REPORT GENERATION Solubility Browser The ProfileLynx Results Browsers contains up to 3 sections: 1.Results Table 2.Chromatogram 3.Calibration Curve Pass/Fail indicator column and user selected highlighted flags allow fast review of the data. Chromatogram is interactive for manual integration if needed. Microsomal Stability Browser LogP/LogD Browser ProfileLynx ph Stability Browser Other Assays Supported - Protein Binding (plate or column) - Membrane Permeability (PAMPA, Caco-2, etc.) - Chromatographic Hydrophobicity Index (CHI) - Immobilized Artificial Membrane 2011 Waters Corporation 25
ProfileLynx View by Experiment Single browser to review all experiments 2011 Waters Corporation 26
Discovery ADME Workflow Using QuanOptimize, ProfileLynx & SDMS Sample Formatting Perform ADME stability, solubility etc. Method Development Run Samples QuanOptimize QuanOptimize QuanOptimize Quantify Data Generation QuanOptimize ProfileLynx OpenQuan Report Results ProfileLynx and NuGenesis SDMS 2011 Waters Corporation 27
14 C Structure ID Metabolism Drug-Drug Interaction Candidate Selection & Confirmation Phase 1 GLP Toxicity 28 days/2 species Single dose toxicity Repeated dose toxicity 2011 Waters Corporation 28
Sensitivity Needs Increasing Low Exposure Drugs Micro Sampling Inhaled Products pg/ml sensitivity common place Low sample volumes from mice / tail bled rats Dried blood spot samples 2011 Waters Corporation 29
Designed to deal with problems associated with a larger sampling orifice Electric Field Diffuse Ion Cloud Maximising signal Maximising robustness 2011 Waters Corporation 30
SYNAPT G2-S 2011 Waters Corporation 31
Synapt G2-S Quantitation Compound name: Propanolol Correlation coefficient: r = 0.999771, r^2 = 0.999542 Calibration curve: 1.9722 * x + 0.318197 Response type: Internal Std ( Ref 1 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None Blank 180 160 2.5pg/mL 25ng/mL 140 120 Response 100 80 60 40 20 2.5pg/mL -0 Conc -0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 30x improvement in exact mass sensitivity 2011 Waters Corporation 32
G2-S Sensitivity Product Ion MS E 10µM 1µM 100nM 10nM Confident Structural elucidation can be performed down to 1000- fold dilutions using nondirected MS E fragmentation spectra 2011 Waters Corporation 33
Waters Regulated Bioanalysis System Solution Sample Preparation Solutions Best in class ACQUITY UPLC I-Class Fast, robust method development to high throughput screening Xevo TQ-S Most sensitive MS UNIFI Compliant, Interactive workflow-driven data platform 2011 Waters Corporation 34
Ultra High Sensitivity? UPLC/MRM of Verapamil (solvent standard) using an ACQUITY prepared for trace analysis 100 6 replicates RSD<20% 220 Zeptomoles 100 % 100 0.83 2fg 0.1fg 0.83 of Verapamil % Solvent 0 Time 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 % Blank 0 Time 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 0 Time 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 2011 Waters Corporation 35
Assay Robustness 10ul loop injection 0.5pg/ul Verapamil in human plasma/acn_2 TC8_100721_1135 100 >4000 on column injections RSD of peak areas < 5% 0.83 MRM of 1 Channel ES+ 455 > 165 (Verapamil) 2.66e7 Plasma injection 4000 Plasma injection 2000 Plasma injection 1 % Verapamil, 0.5pg/µL spiked into supernatant from 2:1 ACN:Plasma protein precipitation. 10µL injections ACQUITY BDH 2.1x 50 0 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00 1.05 1.10 1.15 1.20 1.25 Time 2011 Waters Corporation 36
Metabolites In Safety Testing In general, these metabolites are of interest if they account for plasma levels >10% of total systemic exposure to drug related material, measured as area under the curve (AUC) at steady state. 2011 Waters Corporation 37
Simultaneous MRM and Full Scan Detection MRM of Parent Metabolites Full Scan of Metabolites 2011 Waters Corporation 38
Develop Theoretical MRMs to Investigate MSMS of Parent Compound Identify Product Ions From Parent Compound Select Metabolites from list of probable transformations MS Source and Analyzer Conditions Automatic Product Ion Selection Spectra of Parent Automatically Obtained from MassLynx Calculate MRMs and Write MS Method File imrm Method List of Potential Metabolites from Text File 2011 Waters Corporation 39
% % % % Simple Metabolite Detection and Confirmation 18_4_022 100 % 18 _4 _0 23 4 ( 1. 97 6) 1: Product Ions of 416 ES+ 416.148 > 325.847 (AZ1_Dealkylation) 5.5 6e 5 326 1 00 PIC Spectrum 3 13 2 98 0 m/ z 1 00 1 20 14 0 16 0 1 80 20 0 22 0 2 40 2 60 28 0 30 0 3 20 340 36 0 3 80 4 00 42 0 Dealkylation 340 1: MRM of 1 Channel ES+ 18_4_022 416.148 > 325.847 (AZ1_Dealkylation) 5.66e6100 % 18_ 4_ 023 3 (1.871) 2 : Product I ons of 428 ES+ 428.184 > 325.829 (AZ1_Demethylation) 3. 92 e4 326 1 00 PIC Spectrum 14 7 176 23 5 0 m /z 10 0 12 0 140 1 60 1 80 20 0 22 0 24 0 260 2 80 3 00 3 20 34 0 36 0 380 4 00 4 20 Demethylation 340 2: MRM of 1 Channel ES+ 428.184 > 325.829 (AZ1_Demethylation) 3.58e6 [Benefits] Metabolites detected with maximum sensitivity Spectra acquired in the time scale of UPLC peak Data acquired in one analytical run, no need for confirmatory experiment 0 18_4_025 100 0.50 1.00 1.50 2.00 2.50 18 _4 _0 25 5 ( 2. 14 0) 3: Product Ions of 442 ES+ 442.2 > 325. 847 ( AZ1_ Parent) 8.9 8e 6 3 26 1 00 PIC Spectrum Time 3: MRM of 1 Channel ES+ 18_4_022 442.2 > 325.847 (AZ1_Parent) 1.08e8 100 Dosed Compound 0 0.50 1.00 1.50 2.00 2.50 Time 5: MRM of 1 Channel ES+ 458.195 > 339.845 (AZ1_Hydroxylation) 3.77e5 18 _4 _0 23 3 ( 2. 07 8) 5: Product Ions of 458 ES+ 458.195 > 339.845 (AZ1_Hydroxylation) 1.9 2e 5 326 1 00 PIC Spectrum 3 40 % 425 313 0 m/ z 1 00 12 0 14 0 16 0 18 0 2 00 2 20 2 40 26 0 28 0 30 0 3 20 3 40 3 60 38 0 40 0 42 0 44 0 4 60 MRM 442.2>326 % 340 354 315 0 m/ z 1 00 12 0 1 40 1 60 18 0 2 00 2 20 24 0 2 60 28 0 30 0 3 20 34 0 36 0 3 80 40 0 42 0 4 40 46 0 Hydroxylated Metabolite MRM 458.2>340 0 Time0 Time 0.50 1.00 1.50 2.00 2.50 0.50 1.00 1.50 2.00 2.50 2011 Waters Corporation 40
Work-flow Oriented System Solutions Increasing Regulatory Demands Quality and Consistency of Results Evolving Analytical Demands 2011 Waters Corporation 41
UNIFI: Scientific Information System Single Platform Multiple Techniques Elegant & Simple Secure & Compliant Future Proof Unites our endto-end system solution One software for Chromatography & MS Workflow based design adjusts to roles and capabilities of users Compliant and validated for your environment Will grow with your needs 2011 Waters Corporation 42
Pharmacology, Biomarker Metabolism Supporting Studies Candidate Selection & Confirmation Phase 1 Phase 2 Phase 3 Single dose toxicity Submit IND Submit NDA Repeated dose toxicity Supporting Studies 2011 Waters Corporation 43
Ion Mobility Separation 2011 Waters Corporation 44
An added dimension of separation with Synapt HDMS E 2011 Waters Corporation 45
Metabolomic Biomarker Analysis Targeted Chemspider search with >22 million compounds 2011 Waters Corporation 46
Global Lipid Profiling 2011 Waters Corporation 47
Targeted Metabolite Biomarker Analysis Normalized Conc (ng per sample) x 10000 50 45 40 35 30 25 20 15 10 5 0 MTC MTln3 Absolute quantitation of TCA Cycle intermediates with internal standards 2011 Waters Corporation 48
Peptide Biomarker Analysis Amyloid β 0.1 ng/ml Oasis (MCX µ-elution), UPLC, Xevo TQ-S Sensitive, Robust, meets assay requirements Evolving analytical demands 2011 Waters Corporation 49
Drug Development Process Drug Metabolism 14 C Structure ID Metabolism Drug-Drug Interaction Pharmacology, Biomarker Dose Ranging Study Pre-clinical testing R&D - 18 months Clinical R&D - 5 years Candidate Selection & Confirmation Phase 1 Phase 2 Phase 3 PK/PD & ADME GLP Toxicity 28 days/2 species Single dose toxicity Submit IND Proof of Concept PK/PD Submit NDA Toxicity (non-glp) Repeated dose toxicity Xevo G2 QTof Synapt G2-S Xevo TQ(S) MS 2011 Waters Corporation 50
Questions? Thank You 2011 Waters Corporation 51