PEAQ DSC and PEAQ ITC - atomikateknik.com · © Malvern Panalytical MICROCAL PEAQ ITC MicroCal PEAQ...
Transcript of PEAQ DSC and PEAQ ITC - atomikateknik.com · © Malvern Panalytical MICROCAL PEAQ ITC MicroCal PEAQ...
© Malvern Panalytical
PEAQ ITC AND MICROCAL DSC
© Malvern Panalytical
MICROCAL PEAQ ITC
MicroCal PEAQ ITC (Semi-automated) MicroCal PEAQ ITC Automated
© Malvern Panalytical
Interaction Analysis Stability ProfilingBioparticle
Characterization
Solutions for Biosciences
Confirm affinity and
function
Understand solution
behaviour and aggregation
propensity
Understand critical
degradation pathways
Develop robust
formulations
Extend sub-visible range
Identify contaminants
Biotherapeutics, exosomes,
viruses and vaccines
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OVERVIEW
• What is calorimetry
• Isothermal Calorimetry (iTC)
• Differential Scanning Calorimetry (DSC)
© Malvern Panalytical
WHAT IS CALORIMETRY?
Science of measuring the
heat of chemical reactions or
physical changes.
Calorimetry
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›CALOR = heat
›METRUM = measure
›Measure the heat (generated or absorbed)
›Joule = J
›Calorie = cal
› 1 cal = 4.184 J
Calorimetry
© Malvern Panalytical
• Native molecules in solution (biological relevance)
• Very sensitive to accomodate range of affinities
WHY MICROCALORIMETRY?
Label-freeBroad dynamic
rangeEase-of-use
• Direct measurement of heat change (ITC)
• Direct measurement of melting transition temperature to predict thermal stability (DSC)
• No labeling or immobilzation
• No assay development
• Wide range of solvent/buffer conditions
Information rich
• All binding parameters in a single ITC experiment:
AffinityStoichiometryEnthalpyEntropy
0 1 2
-12
-9
-6
-3
0
Xt/Mt
ND
H, k
cal/m
ole
of in
ject
ant
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Two major techniques
Microcal PEAQ DSC automated
Differential scanning calorimetry (DSC) Isothermal titration calorimetry (ITC)
MicroCal PEAQTM ITC
MicroCal
PEAQ ITC
Automated
Microcal PEAQ DSC
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OVERVIEW
• What is calorimetry
• Isothermal Calorimetry (iTC)
• Differential Scanning Calorimetry (DSC)
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WITH ISOTHERMAL TITRATION CALORIMETRY YOU CAN…
• Get quick KDs for secondary screening/hit validation
• Measure target activity (Stoichiometry, active concentration)
• Confirm drug binding to target
• Use thermodynamics to guide lead optimization
• Characterize mechanism of action
• Measure enzyme kinetics
© Malvern Panalytical
HOW DO THEY WORK?
Reference Calibration Heater
Cell Main Heater
Sample Calibration Heater
DP
DT
Sample The DP is a measured power differential between
the reference and sample cells to maintain a zero
temperature between the cells
DT~0DP = Differential power
∆T = Temperature difference
Reference
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PERFORMING AN ITC ASSAY
•“Ligand” in syringe
•“Macromolecule” in sample cell
Reference cell Sample cell
Syringe
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S R
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Reference power
Reference power supplied to the reference cell
1
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Reference power
Reference power supplied to the reference cell activates feedback to sample cell
12
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Reference power
How much energy needs to be applied to the sample cell in order to get zero output
from Peltier element = same temperature in reference and sample cell
3
The signal we
see, DP is this
energy in
uCal/sec
= 0
© Malvern Panalytical
Reference power
An exothermic reaction in the sample cell will cause a temperature offset, activating
the Peltier sensor. The feedback is regulated accordingly until zero output.
4= 0
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Reference power
After reaching equilibrium, the system relaxes to reference power level and is ready
for the next injection
5= 0
© Malvern Panalytical
Compound – in syringeProtein target in ITC cell
ITC – BEFORE TITRATION
© Malvern Panalytical
Compound in syringe
Protein target in cell
Protein target-ligand complex
As the first
injection is made,
the injected
compound
begins to bind to
the target protein.
TITRATION BEGINS: FIRST INJECTION
© Malvern Panalytical
The signal
returns to
baseline before
the next
injection.
FIRST INJECTION READY: RETURN TO BASELINE
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As a second
injection is
made, again the
injected
compound binds
to the target.
SECOND INJECTION
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Signal again
returns to
baseline before
next injection.
SECOND INJECTION READY: RETURN TO BASELINE
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As the injections
continue, the
target becomes
saturated with
compound, so less
binding occurs and
the heat change
starts to decrease.
INJECTIONS CONTINUE…
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As the injections
continue, the
target becomes
saturated with
compound, so less
binding occurs and
the heat change
starts to decrease.
INJECTIONS CONTINUE…
© Malvern Panalytical
When the target
is saturated with
compound, no
more binding
occurs.
END OF TITRATION
© Malvern Panalytical
Raw data
ITC EXPERIMENTAL PRINCIPLE
Reference cell Sample cell
Syringe
In a single ITC experiment you get…
▪ Affinity – strength of binding
▪ Binding mechanism – thermodynamics describes
the driving forces of interaction
▪ Stoichiometry - number of binding sites
Affinity Binding
mechanism Stoichiometry
© Malvern Panalytical
BASICS OF AN ITC EXPERIMENT
Integration of heats are used to extract affinity (KD), stoichiometry (N)
and binding enthalpy (DH) using appropriate binding model
Universal technique based on heat detection
-4
-2
0
0 0.5 1.0 1.5 2.00.0 0.5 1.0 1.5 2.0
-4
-2
0
Molar Ratio
kca
l/m
ole
of i
nje
ctan
t
DH
N
KD
kcal
mo
l-1o
f in
ject
ant
Molar ratio
µca
l s-1
Time ->
© Malvern Panalytical
THE ENERGETICS
-14
-12
-10
-8
-6
-4
-2
0
kcal/m
ole
of in
jecta
nt
0 1 2 3 4
› The same affinity and
stoichiometry, but different
enthalpy (heat)
› Different binding mechanisms
Ligand A into
compound X
Ligand B into
compound X
Molar ratio
© Malvern Panalytical
THE ENERGETICS
DG = RT ln KD
DG = DH –TDS
∆G = Gibbs free energy
∆ H = Enthalpy
∆ S = Entropy
R = Gas constant = 1.985 cal K-1 mol-1
T = Temperature in Kelvin = 273.15 + t 0C
KD = Affinity (Diss Constant)
› ΔH, enthalpy is indication of changes in
hydrogen and van der Waals bonding
› -TΔS, entropy is indication of changes
in hydrophobic interaction and/or
conformational changes
© Malvern Panalytical
CHALLENGES WITH ITC
• Concentrations
• Buffers/Sample preparation
• Examples
© Malvern Panalytical
ASSESSMENT OF PROTEIN QUALITY: MICROCAL™ ITC200 SYSTEM
•100% of Batch 1 protein activebased on stoichiometry
Presented by L.Gao (Hoffmann-La Roche), poster at SBS 2009
Peptide binding to protein Batch #1 Peptide binding to protein Batch #2
›23% of Batch 2 protein active based on stoichiometry
© Malvern Panalytical
Antibody/Antigen
Same N in both
experiments =
can compare data
30 uM bi-valent Ab in syringe, 4 uM antigen in cell
© Malvern Panalytical
POOR SAMPLE PREPARATION LEADS TO POOR DATA
•The data shown here shows
before and after dialysis
•The large peaks were due
to differences in the NaCl
concentration between
buffers
0 20 40 60 80 100 120 140 160 180
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
without dialysis
with dialysis
Time (min)
µca
l/se
c
With
dialysis
Without
dialysis
© Malvern Panalytical
The ligand
Dilute an aliquot of the ligand stock solution containing dimethylsulfoxide
(DMSO) with the dialysate and then…
The protein
Add a corresponding amount of DMSO to the protein solution
SAMPLE PREPARATION (SOLVENTS, DETERGENTS)
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LIGAND PREPARATION FROM DMSO STOCKSAMPLE PREPARATION
5 mM ligand
in 100% DMSO50 µl
Dialysate
buffer950 µl
250 µM ligand
in 5% DMSO
© Malvern Panalytical
MATCH DMSO IN THE PROTEIN SOLUTIONSAMPLE PREPARATION
DMSO
50 µl
25 µM dialyzed
protein950 µl
1 ml of 23.75 µM
protein in 5% DMSO
© Malvern Panalytical
DMSO MISMATCHSAMPLE PREPARATION
LARGE HEATS FROM DMSO DILUTION, IF BUFFERS ARE NOT MATCHED
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00
Time (min)
0.5 cal/sec
Buffer into buffer
5% DMSO into 5% DMSO
5% DMSO into 4.5% DMSO
5% DMSO into 4 % DMSO
© Malvern Panalytical
SOLVENT SCOUTING
Dialysate
buffer
DMSO
Dialysate buffer 4.8% DMSO
Dialysate buffer 4.9% DMSO
Dialysate buffer 5.0% DMSO
Dialysate buffer 5.1% DMSO
Dialysate buffer 5.2% DMSO
250 µM ligand in
“5%” DMSO
In the syringe
© Malvern Panalytical
SOLVENT SCOUTING
Dialysate
buffer
DMSO
Dialysate buffer 4.8% DMSO
Dialysate buffer 4.9% DMSO
Dialysate buffer 5.0% DMSO
Dialysate buffer 5.1% DMSO
Dialysate buffer 5.2% DMSO
250 µM ligand in
“5%” DMSO
In the syringe
© Malvern Panalytical
ITC DATA: RESOLUTION AND CONCENTRATIONS
DEMO DATA:
STABILE PROTEIN (GAL3)
LMW LIGAND
HEPES, 5% DMSO
FIRST DATA SET
2ND DATA POINT
WHY?
0.0 0.5 1.0 1.5
-26.0
-24.0
-22.0
-20.0
-18.0
-16.0
-14.0
-12.0
-10.0
-8.0
-6.0
-4.0
-2.0
0.0
-1.00
-0.80
-0.60
-0.40
-0.20
0.00
0 10 20 30 40
Time (min)
µca
l/se
c
230 uM protein in syringe
33 uM LMW ligand in cell
Molar Ratio
kca
l m
ol-1
of
inje
cta
nt
!
© Malvern Panalytical
ITC DATA: RESOLUTION AND CONCENTRATIONS
SECOND DATA SET
REPLACED PLUNGER TIP
DEEP CLEAN
2ND DATA POINT STILL OFF
0.0 0.5 1.0 1.5
-24.0
-22.0
-20.0
-18.0
-16.0
-14.0
-12.0
-10.0
-8.0
-6.0
-4.0
-2.0
0.0
-1.00
-0.80
-0.60
-0.40
-0.20
0.00
0 10 20 30 40
Time (min)
µca
l/se
c
Molar Ratio
kca
l m
ol-1
of
inje
cta
nt
!
© Malvern Panalytical
0.0 0.5 1.0 1.5
-28.0
-26.0
-24.0
-22.0
-20.0
-18.0
-16.0
-14.0
-12.0
-10.0
-8.0
-6.0
-4.0
-2.0
-1.20
-1.00
-0.80
-0.60
-0.40
-0.20
0.00
0 10 20 30 40
Time (min)
µca
l/se
c
Molar Ratio
kca
l m
ol-1
of
inje
cta
nt
CONCENTRATIONS
THIRD SETSTILL THERE, ITS REAL!
© Malvern Panalytical
0.0 0.5 1.0
-26.3
-23.9
-21.5
-19.1
-16.7
-14.3
-11.9
-9.6
-7.2
-4.8
-2.4
0.0
2.4
-0.14
-0.10
-0.05
0.00
0 10 20 30 40 50 60
Time (min)
µca
l/se
c
50 uM protein in syringe
9 uM LMW ligand in cell
Data: D139Gal3zz_NDH
Model: TwoSites
Chi^2 = 1.860E5
N1 2.35 ±0.00832 Sites
K1 8.18E9 ±3.88E9 M-1
DH1 -8671 ±53.4 cal/mol
DS1 16.6 cal/mol/deg
N2 6.39 ±0.242 Sites
K2 5.41E6 ±2.61E6 M-1
DH2 -945.6 ±50.9 cal/mol
DS2 27.7 cal/mol/deg
Molar Ratio
kca
l m
ol-1
of
inje
cta
nt
ITC DATA: RESOLUTION AND CONCENTRATIONS
LOWER CONCETRATION
LOWER INJECTION VOLUMES
LOW FEEDBACK
MORE DATA POINTS
HIGHER RESOLUTION
SECOND BINDING SITE OR
LIGAND MIXTURE
© Malvern Panalytical
DNA INTO PROTEIN
• Aim to investigate N and affinity of different protein constructs
• The higher protein number, the larger construct
• 80 µM of DNA in syringe, 100 µM of protein in the cell
• Samples direct from production (no dialysis…)
© Malvern Panalytical
DNA INTO PROTEIN
Protein 5 Protein 6 Protein 9
Filename Temperature (°C) [Syr] (M) [Cell] (M)
Ligand in
Cell Control Type N (sites) KD (M)
∆H
(kcal/mol) ∆G (kcal/mol) -T∆S (kcal/mol) Offset (kcal/mol)
DNA into Protein5 30 8.00E-05 1.00E-04 Yes Fitted Offset 18.7 1.83E-05 -2.71 -6.57 -3.86 -0.37
DNA into protein6 30 8.00E-05 1.00E-04 Yes Fitted Offset 200 2.11E-03 -26.2 -3.71 22.4 36.8
DNA into protein9 30 8.00E-05 1.00E-04 Yes Fitted Offset 14.3 1.76E-05 -1.97 -6.6 -4.62 -4.05
© Malvern Panalytical
MICROCAL PEAQ ITC
• The latest and 5th generation ITC from
MicroCal• Guided workflows, experimental design
software and fully integrated wash module
for consistently high quality data
• Robust and rapid data analysis
• Improved signal to noise (SW and HW)
© Malvern Panalytical
WASH MODULE • Easier to use
Clearly labeled
tubing and easy to
use fittings
Larger reagent and
waste containers
Automated ‘deep
clean’ at
elevated
temperature
© Malvern Panalytical
MAINTENANCE ALERTS
• Links to built-in videos to demonstrate how to perform straightforward maintenance tasks
Consistent, high quality data
Click on alert for guidance
© Malvern Panalytical
NEW DATA ANALYSIS SOFTWARE
• Automated data qualification
• Robust automated data analysis
• Robust batch analysis of multiple data sets
• Multiple inbuilt tools to graphically visualize
the data
• New features to support common
applications such as SAR
© Malvern Panalytical
OVERVIEW
• What is calorimetry
• Isothermal Calorimetry (iTC)
• Differential Scanning Calorimetry (DSC)
© Malvern Panalytical
DIFFERENTIAL SCANNING CALORIMETRY (DSC)
• Characterize and select the most stable protein or bio-therapeutic candidate
• Optimize expression, purification and manufacturing conditions in days
• Rapidly and easily determine optimum conditions for liquid formulations
Native Unfolded
© Malvern Panalytical
MELTING TEMPERATURE (TM) - INDICATOR OF THERMAL STABILITY
Tm is the thermal transition midpoint
›50% native / 50% unfolded
Stabilizing conditions and/or event
›Both intrinsic and extrinsic
›Unfold at higher temperature
Destabilizing conditions and/or events
›Both intrinsic and extrinsic
›Unfold at lower temperature
Differential Scanning Calorimetry
Native
Unfolded
© Malvern Panalytical
DSC EXPERIMENTAL PRINCIPLEPROTEIN UNFOLDING
FOLLOWED THROUGH HEAT CHANGES
30 40 50 60 70 80 90
0
2
4
6
8
10
12
14
Cp
(kca
l/m
ole
/o C)
Temperature (C)
Folded Unfolded
T=20°C T=90°C
2
6
10
14
Cp
(kca
l/m
ole
/o C)
30 40 50 60 70 80 90
oTemperature(C)
D
© Malvern Panalytical
APPLICATIONS FOR STABILITY DETERMINATION USING TM SHIFT ANALYSIS
• Control (e.g. native)
• Mutant form
Potentially shows:
• Post-translational changes
• Alternative buffer composition
• Long term storage effects
© Malvern Panalytical
RESULT: INCREASES IN THERMAL STABILITY OBSERVED BY TM
SHIFTING
© Malvern Panalytical
MICROCAL VP-DSC SYSTEM
▪ Directly measures the heat of binding (enthalpy, ΔH) and transition midpoint (Tm)
▪ Single sample (130ul cell)
▪ Manual sample loading
▪ Unattended operation
▪ Tantalum Cell
▪ Coin shaped cell
▪ Forward and back scans
© Malvern Panalytical
MICROCAL VP-CAPILLARY DSC• Directly measures the heat of binding (enthalpy, ΔH) and transition
midpoint (Tm)
• Single sample (500ul cell)
• Manual sample loading
• Unattended operation
• Tantalum Cell
• Capillary Cell
• Full automation upgrade
© Malvern Panalytical
MICROCAL VP AUTO CAP-DSC SYSTEM
▪ Directly measures the heat of binding (enthalpy, ΔH) and transition midpoint (Tm)
▪ Single sample (130ul)
▪ Automated sample loading
▪ Unattended operation
▪ Tantalum cell
▪ Capillary cell
▪ 6 x 96 well plates (4oC storage)
▪ Automated washing
© Malvern Panalytical
EXAMPLE 1: THE USE OF DSC TO AID IN THE SELECTION OF
ANTIBODY MUTANTS
MOST STABLE ANTIBODY CONSTRUCTS IDENTIFIED
• Stability of each domain can be
assessed
• Minor differences in primary sequence
can have a big impact on antibody
stability
• The least stable expressed poorly and
quickly formed high
MW aggregates
Demarest et al, Application note
© Malvern Panalytical
THE SOLUTION: IDENTIFY STABILIZING ELUTION CONDITIONS
USING DSC
• DSC identified the most
stabilizing elution conditions
• This allowed increased functional
loading capacity since mAb is
stabilized
• Starting buffer Citrate pH 3.5
• Optimum buffer Citrate plus
Mannitol pH 3.5 = Highest Tm
P. Acharya, application note
© Malvern Panalytical
ANTIBODY STABILITY 2
© Malvern Panalytical
ANTIBODY STABILITY 3
DSC unfolding curves of the BIIB7 antibody in both the IgG1 and IgG4 formats
© Malvern Panalytical
ANTIBODY FOLDING AND TM• The domains involved in the pH-sensitive transition were completely unfolded at pH
4.5, based on the structural data obtained using CD
• This demonstrates how DSC can be important not only for understanding the stability
of folded domains, but their folding status as well
© Malvern Panalytical
DSC AND BINDING
© Malvern Panalytical
TM INCREASE WITH LIGAND CONCENTRATION
RNase plus increasing concentrations of 2’ CMP
© Malvern Panalytical
THE PROCESSED DATA – AFFINITY ESTIMATES
0 20 40 60 80 100
-5
0
5
10
15
20
25
Rnase only - no ligand
Rnase + Phosphate"
RNase + 3'CMP""
RNAse + 2'CMP"
Rnase only - no ligand
Rnase + Phosphate"
RNase + 3'CMP""
RNAse + 2'CMP"
Rnase only - no ligand
Rnase + Phosphate"
RNase + 3'CMP""
RNAse + 2'CMP"
Cp
(kca
l/m
ole
/oC
)
Temperature (oC)
KB = 1/KD
© Malvern Panalytical
• Three lots manufactured at different sites
• DSC verifies no difference in stability and solubility between lots
Jiang and Nahri, American Pharmaceutical
Review, 2006 (on-line)
EASILY ASSESS BIO-COMPARABILITY WITH DSC
© Malvern Panalytical
TEŞEKKÜRLER