RapidFire High-throughput MS technology. Enhancing Drug Discovery Turning Mass Specs into Plate Readers
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1 RapidFire High-throughput MS technology Enhancing Drug Discovery Turning Mass Specs into Plate Readers 1
2 Drug Discovery with RapidFire Target Selection High-throughput Target Screening (HTS) Lead Optimization Preclinical Testing Clinical Trials Genomics Proteomics Metabolomics Lipidomics Animal models Knockouts Label-free HTS using native substrate Secondary screen Selectivity screen Med Chem / SAR ADMET PK/PD Toxicology PK/PD TDM DDI Met Stab Permeability Protein binding Identify the best hits: Eliminate HTS compromises Select the best leads: Data-driven lead selection Eliminate compromises when designing experiments 2
3 RapidFire Mass Spectrometry Fast autosampler & SPE system Replaces LC in LC/MS Integrated, automated, micro-scale solid-phase extraction Integrates with standard ESI MS instruments cycle time: 5 10s/sample Compatible with biological matrices Microsomal preparations Cell culture supernatants Tissue extracts Plasma
4 SPE-MS/MS Analysis Mass spec Aqueous wash Organic reverse elution waste
5 RapidFire Data 96-well plate analyzed in <12 minutes IS data Sample data 5
6 RapidFire Cycle Sip/Equilibrate Sip Elute Load Standard 96 or 384-well plates Sample interface (sipper) Washed with aqueous and organic solvents Optical sip sensor o wells missed (even if half full) 6
7 RapidFire Cycle Load/Wash Sip Elute Load 10-µL injection loop onto 4-µL RapidFire cartridge o offline sample preparation SPE purification by proprietary cartridge (variety of packing beds) of quenched assays Cartridge changer (6 slots) Automatic or programmed switch 24-hour unattended runs Facilitates assay development Packing Material C4, C8, C18 Phenyl, Cyano HILIC Client specified 1.4 cm 7
8 RapidFire Cycle Elute/Clean Sip Elute Load Compatible with any ESI or APCI flow injection analysis MS MS at 5-10 seconds/sample Micro-scale fluidic path Baseline resolution PEEK minimizes carryover 8
9 RapidFire - Automation & Efficiency Acquisition Software Features Sequence-based protocol injections from plates in any well order Multiple MS methods per plate Enter all pre-developed parameters at once for full stack of plates (7000 samples/run) Barcode scanner 24-hour unattended operation Active monitoring Error flag upon suspension 9
10 RapidFire Integrator - Data Analysis & Output Proprietary, intuitive data analysis software Outputs results as text files compatible with standard databases 10
11 RapidFire Integrator Software Internal Standard (d4-1 OH-midazolam) A B C D E F G H Product (1 OH-midazolam) A B C D E F G H Turning a mass spectrometer into a plate reader
12 RapidFire applications for in vitro ADME (absorption-distribution-metabolism-excretion) 12
13 RapidFire for in vitro ADME assays Strong results correlation with traditional technologies CYP inhibition (drug-drug interaction) Reversible and Time-dependent Metabolic stability HLM or S9 Permeability cell lines (Caco-2) and/or PAMPA P-glycoprotein inhibition Plasma protein binding HSA, brain, microsomal binding 13
14 IC 50 (um) by Traditional RF-MS Summary of Analytical Methods Comparison 1000 R 2 = Direct CYP Inhibition: Comparable results obtained >20-fold faster analysis 8 different isoforms/probe pairs IC50 (um) by BioTrove CYP1A2 CYP2B6 CYP2C8 CYP2C9 CYP2C19 CYP2D6 CYP3A4 Caco-2 Permeability: Comparable results obtained nearly 10x faster with RF-MS LC-MS RF-MS Analysis Time 20 hours 2.5 hours IC IC 50 (um) 50 (um) by by Traditional LCMS Gentest Linearity R > 0.99 R > 0.98 LLOQ 5 nm 5 nm Carry-over < 0.3% < 0.3% Generic method applicability Generic LC method Generic SPE method 14
15 RapidFire 360 High Resolution System Eliminates bottlenecks in high-throughput MS The most efficient analysis available o MRM method development Fully integrated sample preparation Walk-away automation
16 RF360 Removes the MRM Bottleneck Traditional MS/MS RF360 Run Assay Run Assay 4-10 min/ analyte 1-3/sample (15-45 min/ analyte) QqQ method development Data Acquisition Generic Methods & Full MS Scan Data Acquisition Data Analysis 6-10 sec/sample (1-2 min/analyte) Data Analysis
17 RF 360 Data Microsomal Stability Assay with Midazolam TIC Parent (Midazolam) Metabolite (Hydroxymidazolam) 0 min 5 min 10 min 20 min 30 min 60 min Extraction and peak integration of analytes from TIC is fully automated Internal Standard (Bupivicaine) 17
18 Metabolite Analysis Data 18
19 High-throughput TOF Data Analysis monoxidation dioxidation demethylation deethylation reduction desatuation glucuronadation sulphation acetylation Import mass table or formulas Select metabolites of interest Obtain fully-integrated peak areas 19
20 Prod/IS, AUC Metabolite Analysis Results - Midazolam Midazolam Time, min Midazolam Oxidation Cl F Cl F HO
21 Prod/IS, AUC Prod/IS, AUC Metabolite Analysis Results - Chloropromazine Chloropromazine Chloropromazine Metabolites oxidation demethylation 2 demethylations Time, min Time, min H H 2 Cl Cl Cl S S S Cl OH S
22 Reducing Bottlenecks in ADME Sample Analysis using Solid Phase Extraction with a Quadrupole Time-of-Flight Mass Spectrometer Traditional LC-MS Workflow 38.5 hr 0.5 hr 12 hr 24 hr 2 hr Samples Received Batch Creation Analyte Tuning RapidFire 300 MS QQQ Work Flow Samples Received 16.5 hr 0.5 hr 12 hr 2 hr 2 hr Sample Analysis Data Delivery Data Analysis Data Delivery Adapted from oral presentation by Panos Hatsis, ovartis, 58 th ASMS Conference on Mass Spectrometry and Allied Topics, May 23-27, 2010 RapidFire 360 TOF Work Flow Samples Received 4.5 hr Data Delivery
23 Resolves Bottlenecks Improved Data Quality: Throughput allows for Multiple concentrations: run full IC 50 s Multiple time points Avoids surrogate substrates, indirect and coupled assays Maximizes Productivity: Fastest data turnaround 10x data generated per FTE compared to LC-MS Rapid assay development in HTS faster time to answer Strong Correlation with Traditional Technologies: LC-MS/MS: CYP450 inhibition, metabolic stability, etc Optical-probe or radioactivity-based detectors
24 Advantages of Lower Operational Costs: Solvents: 6 ml/min = 0.25 ml (~2 /sample) Cartridge: $200/cartridge or ~ 3000 samples/cartridge (~7 /sample) Minimal Reagent & Disposal Costs in HTS: Only native substrate & enzyme are required o antibodies, luminescent or radioactive reagents, kits Fits Existing Workflows Designed to operate similar to a plate reader
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