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
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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
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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
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Types of Homogenization
Mechanical Homogenization
Shear force
Potter®
Autogizer®
Acoustical force
(Shock wave)
Covaris®
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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

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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
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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
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New Technology: Adaptive Focused Acoustic (AFA)
Covaris SX2

Isothermal
Frequency 500 KHz
Controlled wave packets
Single sample homogenization
Bench top instrument
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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
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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
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Initial Project
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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
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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)
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in-house engineered devices

suitable stopper
(not available commercially)
tight fit, retains perfectly
PnP arm
can easily pick them
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avoids any aerosol
during
homogenization
in-house engineered devices
multiple masks for different tubes
just one
cooled
rack
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Automated Workflow
Instrument
Arm
Workflow
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…playing Tecan Freedom Evo-Covaris Sx2 Video
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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
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Covaris: program examples
RAT
3.5 cycles
RAT
MOUSE
5 cycles
4 cycles
RAT
6.5 cycles
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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)
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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
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800
1000
Dilution Media Comparison
WHY BUFFER?
9 Interferences:
trans-esterification, microprecipitation, pH shift
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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
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400
450
500
Dilution Factor Comparison
1:3
WHY 1:5?
9 Automation
9 Potential ion
suppression
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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
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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.
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Muscle: methods
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Muscle: results
with collagenase
and 5’21” homogenization
without collagenase
15’ homogenization
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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
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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