PEAQ DSC and PEAQ ITC - atomikateknik.com · © Malvern Panalytical MICROCAL PEAQ ITC MicroCal PEAQ...

PEAQ ITC AND MICROCAL DSC

MICROCAL PEAQ ITC

MicroCal PEAQ ITC (Semi-automated) MicroCal PEAQ ITC Automated

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

OVERVIEW

• What is calorimetry

• Isothermal Calorimetry (iTC)

• Differential Scanning Calorimetry (DSC)

WHAT IS CALORIMETRY?

Science of measuring the

heat of chemical reactions or

physical changes.

Calorimetry

›CALOR = heat

›METRUM = measure

›Measure the heat (generated or absorbed)

›Joule = J

›Calorie = cal

› 1 cal = 4.184 J

Calorimetry

• 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

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

OVERVIEW

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

HOW DO THEY WORK?

Reference Calibration Heater

Cell Main Heater

Sample Calibration Heater

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

PERFORMING AN ITC ASSAY

•“Ligand” in syringe

•“Macromolecule” in sample cell

Reference cell Sample cell

Syringe

Reference power

Reference power supplied to the reference cell

Reference power

Reference power supplied to the reference cell activates feedback to sample cell

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

The signal we

see, DP is this

energy in

uCal/sec

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.

Reference power

After reaching equilibrium, the system relaxes to reference power level and is ready

for the next injection

Compound – in syringeProtein target in ITC cell

ITC – BEFORE TITRATION

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

The signal

returns to

baseline before

the next

injection.

FIRST INJECTION READY: RETURN TO BASELINE

As a second

injection is

made, again the

injected

compound binds

to the target.

SECOND INJECTION

Signal again

returns to

baseline before

next injection.

SECOND INJECTION READY: RETURN TO BASELINE

As the injections

continue, the

target becomes

saturated with

compound, so less

binding occurs and

the heat change

starts to decrease.

INJECTIONS CONTINUE…

As the injections

continue, the

target becomes

saturated with

compound, so less

binding occurs and

the heat change

starts to decrease.

INJECTIONS CONTINUE…

When the target

is saturated with

compound, no

more binding

occurs.

END OF TITRATION

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

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

0 0.5 1.0 1.5 2.00.0 0.5 1.0 1.5 2.0

Molar Ratio

Molar ratio

Time ->

THE ENERGETICS

kcal/m

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

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

CHALLENGES WITH ITC

• Concentrations

• Buffers/Sample preparation

• Examples

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

Antibody/Antigen

Same N in both

experiments =

can compare data

30 uM bi-valent Ab in syringe, 4 uM antigen in cell

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

without dialysis

with dialysis

Time (min)

dialysis

Without

dialysis

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)

LIGAND PREPARATION FROM DMSO STOCKSAMPLE PREPARATION

5 mM ligand

in 100% DMSO50 µl

Dialysate

buffer950 µl

250 µM ligand

in 5% DMSO

MATCH DMSO IN THE PROTEIN SOLUTIONSAMPLE PREPARATION

50 µl

25 µM dialyzed

protein950 µl

1 ml of 23.75 µM

protein in 5% DMSO

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

SOLVENT SCOUTING

Dialysate

buffer

Dialysate buffer 4.8% DMSO

250 µM ligand in

“5%” DMSO

In the syringe

SOLVENT SCOUTING

Dialysate

buffer

250 µM ligand in

“5%” DMSO

In the syringe

ITC DATA: RESOLUTION AND CONCENTRATIONS

DEMO DATA:

STABILE PROTEIN (GAL3)

LMW LIGAND

HEPES, 5% DMSO

FIRST DATA SET

2ND DATA POINT

0.0 0.5 1.0 1.5

0 10 20 30 40

Time (min)

230 uM protein in syringe

33 uM LMW ligand in cell

Molar Ratio

SECOND DATA SET

REPLACED PLUNGER TIP

DEEP CLEAN

2ND DATA POINT STILL OFF

0.0 0.5 1.0 1.5

0 10 20 30 40

Time (min)

Molar Ratio

0.0 0.5 1.0 1.5

0 10 20 30 40

Time (min)

Molar Ratio

CONCENTRATIONS

THIRD SETSTILL THERE, ITS REAL!

0.0 0.5 1.0

0 10 20 30 40 50 60

Time (min)

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

LOWER CONCETRATION

LOWER INJECTION VOLUMES

LOW FEEDBACK

MORE DATA POINTS

HIGHER RESOLUTION

SECOND BINDING SITE OR

LIGAND MIXTURE

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…)

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)

(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

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)

WASH MODULE • Easier to use

Clearly labeled

tubing and easy to

use fittings

Larger reagent and

waste containers

Automated ‘deep

clean’ at

elevated

temperature

MAINTENANCE ALERTS

• Links to built-in videos to demonstrate how to perform straightforward maintenance tasks

Consistent, high quality data

Click on alert for guidance

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

OVERVIEW

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

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

DSC EXPERIMENTAL PRINCIPLEPROTEIN UNFOLDING

FOLLOWED THROUGH HEAT CHANGES

30 40 50 60 70 80 90

Temperature (C)

Folded Unfolded

T=20°C T=90°C

30 40 50 60 70 80 90

oTemperature(C)

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

RESULT: INCREASES IN THERMAL STABILITY OBSERVED BY TM

SHIFTING

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

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

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

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

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

ANTIBODY STABILITY 2

ANTIBODY STABILITY 3

DSC unfolding curves of the BIIB7 antibody in both the IgG1 and IgG4 formats

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

DSC AND BINDING

TM INCREASE WITH LIGAND CONCENTRATION

RNase plus increasing concentrations of 2’ CMP

THE PROCESSED DATA – AFFINITY ESTIMATES

0 20 40 60 80 100

Rnase only - no ligand

Rnase + Phosphate"

RNase + 3'CMP""

RNAse + 2'CMP"

Rnase + Phosphate"

RNase + 3'CMP""

RNAse + 2'CMP"

Rnase + Phosphate"

RNase + 3'CMP""

RNAse + 2'CMP"

Temperature (oC)

KB = 1/KD

• 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

TEŞEKKÜRLER

PEAQ DSC and PEAQ ITC - atomikateknik.com · © Malvern Panalytical MICROCAL PEAQ ITC MicroCal PEAQ...

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