Waters Solutions for DMPK and Biomarker Analysis. Waters User Meeting ASMS 2011 Denver, Colorado. Stephen McDonald

Transcription

1 Waters Solutions for DMPK and Biomarker Analysis Waters User Meeting ASMS 2011 Denver, Colorado Stephen McDonald 2011 Waters Corporation 1

2 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

3 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

4 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

5 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

6 Drug Metabolism PK/PD & ADME Toxicity (non-glp) 2011 Waters Corporation 6

7 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 ng/ml Waters Corporation 7

8 The tools that power the discovery DMPK solution 2011 Waters Corporation 8

9 Nefazodone MS E Analysis 2011 Waters Corporation 9

10 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) x m/z 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

11 Applying Chemical Intelligence 2011 Waters Corporation 11

12 Where Is My Hydroxylation? 2011 Waters Corporation 12

13 Automated Localization of Biotransformation 2011 Waters Corporation 13

14 Accurate Mass Quantification of 50pg/mL in Plasma _19_6_ : TOF MS ES % Time 2011 Waters Corporation 14

15 Accurate Mass To Detect Peak in Complex matrix Da Window Nominal Mass % Da 50mDa Window Accurate Mass % 0 Time Waters Corporation 15

16 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 Response ng/ml Waters Corporation 16

17 Fragment Ions: MS E Elevated Energy Data e % m/z Waters Corporation 17

18 Calibration Lines Using Fragment Ion and Precursor Ion LLOQ = 500pg/mL m/z = (10mDa) Compound name: propranolol high energy Correlation coefficient: r = , r^2 = Calibration curve: * x Response type: External Std, Area Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None Response pg/ml m/z = (10mDa) LLOQ = 500pg/mL Compound name: propranolol high energy Correlation coefficient: r = , r^2 = Calibration curve: * x Response type: External Std, Area Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None Response pg/ml Waters Corporation 18

19 Accessible Quantitative Analysis 2011 Waters Corporation 19

20 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

21 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

22 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 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

23 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

24 OpenQuan - Open Access Optimization and Quantitation Login User name, job ID, Finish Results by 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

25 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

26 ProfileLynx View by Experiment Single browser to review all experiments 2011 Waters Corporation 26

27 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

28 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

29 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

30 Designed to deal with problems associated with a larger sampling orifice Electric Field Diffuse Ion Cloud Maximising signal Maximising robustness 2011 Waters Corporation 30

31 SYNAPT G2-S 2011 Waters Corporation 31

32 Synapt G2-S Quantitation Compound name: Propanolol Correlation coefficient: r = , r^2 = Calibration curve: * x Response type: Internal Std ( Ref 1 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None Blank pg/mL 25ng/mL Response pg/mL -0 Conc x improvement in exact mass sensitivity 2011 Waters Corporation 32

33 G2-S Sensitivity Product Ion MS E 10µM 1µM 100nM 10nM Confident Structural elucidation can be performed down to fold dilutions using nondirected MS E fragmentation spectra 2011 Waters Corporation 33

34 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

35 Ultra High Sensitivity? UPLC/MRM of Verapamil (solvent standard) using an ACQUITY prepared for trace analysis replicates RSD<20% 220 Zeptomoles 100 % fg 0.1fg 0.83 of Verapamil % Solvent 0 Time % Blank 0 Time Time Waters Corporation 35

36 Assay Robustness 10ul loop injection 0.5pg/ul Verapamil in human plasma/acn_2 TC8_100721_ >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 Time 2011 Waters Corporation 36

37 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 Waters Corporation 37

38 Simultaneous MRM and Full Scan Detection MRM of Parent Metabolites Full Scan of Metabolites 2011 Waters Corporation 38

39 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

40 % % % % Simple Metabolite Detection and Confirmation 18_4_ % 18 _4 _ ( ) 1: Product Ions of 416 ES > (AZ1_Dealkylation) 5.5 6e PIC Spectrum m/ z Dealkylation 340 1: MRM of 1 Channel ES+ 18_4_ > (AZ1_Dealkylation) 5.66e6100 % 18_ 4_ (1.871) 2 : Product I ons of 428 ES > (AZ1_Demethylation) e PIC Spectrum m /z Demethylation 340 2: MRM of 1 Channel ES > (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_ _4 _ ( ) 3: Product Ions of 442 ES > ( AZ1_ Parent) 8.9 8e PIC Spectrum Time 3: MRM of 1 Channel ES+ 18_4_ > (AZ1_Parent) 1.08e8 100 Dosed Compound Time 5: MRM of 1 Channel ES > (AZ1_Hydroxylation) 3.77e5 18 _4 _ ( ) 5: Product Ions of 458 ES > (AZ1_Hydroxylation) 1.9 2e PIC Spectrum 3 40 % m/ z MRM 442.2>326 % m/ z Hydroxylated Metabolite MRM 458.2>340 0 Time0 Time Waters Corporation 40

41 Work-flow Oriented System Solutions Increasing Regulatory Demands Quality and Consistency of Results Evolving Analytical Demands 2011 Waters Corporation 41

42 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

43 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

44 Ion Mobility Separation 2011 Waters Corporation 44

45 An added dimension of separation with Synapt HDMS E 2011 Waters Corporation 45

46 Metabolomic Biomarker Analysis Targeted Chemspider search with >22 million compounds 2011 Waters Corporation 46

47 Global Lipid Profiling 2011 Waters Corporation 47

48 Targeted Metabolite Biomarker Analysis Normalized Conc (ng per sample) x MTC MTln3 Absolute quantitation of TCA Cycle intermediates with internal standards 2011 Waters Corporation 48

49 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

50 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

51 Questions? Thank You 2011 Waters Corporation 51