Document 281815
Fully-automated sample preparation and purification of homogenized tissue samples Berlin, 23 february 2009 Claudia Mundi H. Astner, S. Braggio, S. Fontana, R. Longhi Contents Why analyzing tissue samples Approaches to tissue sample preparation Theoretical Principles and Comparison Project Investment & Realization Fully Automated Procedure Workflow Example of Fully Automated Tissue Sample Preparation (watch video) Tuning Setting & Conditions Process Validation Other Applications and Scripts Conclusions Claudia Mundi – Berlin, 23 February 2009 PK study in pre-clinical species… getting the most! SYSTEMIC BLOOD Classical PK parameters Cl, Vdss, F%, T1/2 SYSTEMIC BLOOD fraction unbound LIVER Site of toxicity concentrations Liver/Blood BRAIN Site of action concentrations Brain/Blood BRAIN fraction unbound PORTAL VEIN BLOOD Fraction absorbed, hepatic clearance MUSCLE Tissues distribution Claudia Mundi – Berlin, 23 February 2009 MUSCLE fraction unbound Tissue Preparation Methods Challenges Considerations Studies limited by number and/or size of samples Lack of true standard reference samples Difficult to automate due to nature of matrix Cross-contamination during processing Potentially greater matrix interferences than plasma Total extraction time and final extract volume Extent of manual intervention Cost Target analyte stability during preparation Claudia Mundi – Berlin, 23 February 2009 Types of Homogenization Mechanical Homogenization Shear force Potter® Autogizer® Acoustical force (Shock wave) Covaris® Claudia Mundi – Berlin, 23 February 2009 Enzymatic Digestion Collagenase Proteinase K Mechanical Homogenization (shear force) Tissue Preparation Method POTTER 1.Harvest 2.Weigh 3.Dilution 4.Homogenize 5.Aliquots 6.Extract (7.Evaporate) 8.Reconstitute 9.Analyze by LC-MS-MS AUTOGIZER Advantages Claudia Mundi – Berlin, 23 February 2009 5 samples at one time Partial Automation Autogizer (Tomtec®) Disadvantages Non-specific binding (need for organic solvent) Cross-contamination Bad homogenization for fibrous tissues Potential (thermal) degradation Noisy Claudia Mundi – Berlin, 23 February 2009 Enzymatic Digestion •Enzymes do the work, without mechanical intervention Method •Easily automated, low cost, no cross-contamination 1.Harvest 2.Weigh 3.Enzymatically Digest 4. Aliquots 5.Centrifuge 6.Extract •Issue: Compound Stability, sample prep variation (?) 8.Reconsitute 9.Analyze by LC/MS/MS •Incubate tissue sample with enzyme overnight (7.Evaporate) C. Yu, L. Penn, J. Hollembaek, W. Li, L. Cohen; Anal. Chem., 2004, 76, 1761-1767 Claudia Mundi – Berlin, 23 February 2009 New Technology: Adaptive Focused Acoustic (AFA) Covaris SX2 Isothermal Frequency 500 KHz Controlled wave packets Single sample homogenization Bench top instrument Claudia Mundi – Berlin, 23 February 2009 Adaptive Focused Acoustic (AFA): Benefits Controllable mechanical energy Isothermal Theoretically no-sample prep variation Non-Contact No sample contamination No clean up of instrument post processing Safe - no aerosol risk No sample cross talk No clean-up, nor decontamination Closed vessel No heat passed to sample Automation Challenge Claudia Mundi – Berlin, 23 February 2009 How do we make it compatible with Liquid Handling Systems? Manual Steps for Tissue Samples Manual weighed brain tissue in 15mL plastic tubes Homogenization using Autogizer® Manual dilution with MeOH:H2O (1:1) Manual aliquots of blank and incurred samples Claudia Mundi – Berlin, 23 February 2009 Initial Project Claudia Mundi – Berlin, 23 February 2009 New Equipment big Covaris tube: suitable for CryoPrep 5 2 tube with in-house designed stopper 6 3 small Covaris tube: suitable for CryoPrep 7 8 standard format: scintillation vial 20mL 4 2 6 5 1 7 8 3 1 Claudia Mundi – Berlin, 23 February 2009 Problem Solving Exercises Setting-up problems ; 1. Standard Covaris sample tubes not suitable ; 2. New stoppers needed ; 3. Custom-designed cooled rack needed ; 4. Integration between FreedomEvo and Covaris requested to remove the ; Covaris safety protection… 5. “Challenging” installation of Te-Fill option Programming problems ; 1. ; 2. ; 3. ; “Challenging” setting-up of the Pick&Place gripper Evoware not able to generate automation scripts in .csv format Typical dilution volume (6mL) exceeded the single dilutors capacity (1mL/syringe) 4. Some minor bugs….i.e. Te-Fill initialization failure if not used for a while (2-3 days) Claudia Mundi – Berlin, 23 February 2009 in-house engineered devices suitable stopper (not available commercially) tight fit, retains perfectly PnP arm can easily pick them Claudia Mundi – Berlin, 23 February 2009 avoids any aerosol during homogenization in-house engineered devices multiple masks for different tubes just one cooled rack Claudia Mundi – Berlin, 23 February 2009 Automated Workflow Instrument Arm Workflow Claudia Mundi – Berlin, 23 February 2009 …playing Tecan Freedom Evo-Covaris Sx2 Video Claudia Mundi – Berlin, 23 February 2009 Covaris Parameters Settings 2 fixed alternated runs 20 sec. 50% Duty cycle 10 Intensity 1000 Cycles/Burst 10 sec. 50% Duty cycle 10 Intensity 100 Cycles/Burst Claudia Mundi – Berlin, 23 February 2009 Covaris: program examples RAT 3.5 cycles RAT MOUSE 5 cycles 4 cycles RAT 6.5 cycles Claudia Mundi – Berlin, 23 February 2009 Dilution Alternatives Dilution media (MeOH 50%, water, buffer) Dilution Factor (3-5) Optimized Conditions 9 Buffer: NaCl, KCl, MgCl2, CaCl2, Na2HPO4*7H2O (CSF-like) 9 Dil. Factor 1:5 (4mL per gr of tissue) Claudia Mundi – Berlin, 23 February 2009 Dilution Media Comparison Detected brain conc. (ng/g): Buffer vs Methanol (n= 59) 800 WHY BUFFER? 9 Interferences: trans-esterification, micro-precipitation, pHshift Buffer CSF-like 700 600 500 400 R2= 0.985 300 200 100 0 0 200 400 600 Methanol Claudia Mundi – Berlin, 23 February 2009 800 1000 Dilution Media Comparison WHY BUFFER? 9 Interferences: trans-esterification, microprecipitation, pH shift Claudia Mundi – Berlin, 23 February 2009 Dilution Factor Comparison Detected brain conc. (ng/g): 1:3 vs 1:5 (n= 21) 500 WHY 1:5? 9 Automation 9 Mitigate ion suppression matrix diluted 1:3 450 400 350 300 250 200 R2= 0.996 150 100 100 150 200 250 300 350 matrix diluted 1:5 Claudia Mundi – Berlin, 23 February 2009 400 450 500 Dilution Factor Comparison 1:3 WHY 1:5? 9 Automation 9 Potential ion suppression Claudia Mundi – Berlin, 23 February 2009 Overall Process Validation Detected brain conc. (ng/g): Covaris vs Autogizer (n= 107) 2000 1000 Covaris 500 100 50 R2= 0.972 10 5 1 1 5 10 50 100 Autogizer Claudia Mundi – Berlin, 23 February 2009 500 1000 2000 Liver: methods & results Optimized Conditions 9 Buffer (N2HPO4, KH2PO4, NaCl) 9 Dil. Factor 1:5 (4mL per gr of tissue) 9 Two step dil. & Two step homog. Claudia Mundi – Berlin, 23 February 2009 Muscle: methods Claudia Mundi – Berlin, 23 February 2009 Muscle: results with collagenase and 5’21” homogenization without collagenase 15’ homogenization Claudia Mundi – Berlin, 23 February 2009 Liver & Muscle: alternative procedure 9 Approximately 1.5-2 grams tissue 9 Put in specific “tissue-tubes” and frozen in liquid nitrogen for 2-3 min. 9 Pulverized with Tissue CryoPrep 9 Transferred to a processing tube prior to automated AFA treatments Claudia Mundi – Berlin, 23 February 2009 Help Choosing the Right Script 60 Tecan scripts on demand: just select your 5 digit code A= calibration curve, spike, precipitation, homogenization, weights-dilutionhomogenization, unknown samples aliquots, entire process B= rat brain, rat liver, mouse brain C= MetOH50%, buffer, Te-Fill D= classic tubes with stopper, small covaris tubes, big covaris tubes, scintillation vials E= tare and then weights gross, imports tare and weights gross, imports both tare and gross Claudia Mundi – Berlin, 23 February 2009 Conclusions / Achievements 1 day manageable samples 2000 5 2006 10-15 50 Today 100 Brain Brain Brain Brain •maximized information from a single study •all advantages of automated vs manual process Claudia Mundi – Berlin, 23 February 2009 Liver Muscle Acknowledgements External Collaborations GSK F. Bianchi N. Cesari S. Delle Fratte G. De Nisco V. Hadden B. Perini R. Ricci I. Sartori F. Vinco S. Bauer G.Cimoli/M. Molinaro A. Fusaro N Kruize Claudia Mundi – Berlin, 23 February 2009
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