Better Lives. Better Planet.SM

This presentation is the confidential and copyright work product of Pall Corporation, and no portion of this presentation may be copied, published, performed, or redistributed without the express written authority of a Pall corporate officer.© 2013 Pall Corporation

Kinetics GuideTraining

Anna Sinsigalli, MS

Senior Field Applications Scientist

ForteBio, A Division of Pall Life Sciences

Anna Sinsigalli, MS

Senior Field Applications Scientist

ForteBio, A Division of Pall Life Sciences

2

Kinetics Background

3

Kinetics 101

A ‘classic’ 1:1 binding curve

4

Kinetics 101

� So what is happening at each step in the Sensorgram?

– During association, BOTH on and off processes are occurring at the same time. This is VERY important to understand, since the binding rate you see here is the net difference of the 2 processes. We call this the observed binding rate or kobs.

– During association, the concentration of analyte in solution is kept constant by the stirring. Again, this is important since if the concentration reduced, then the kobs would go down.

– Also during association, the concentration of free ligand on the surface is going down as more complex is formed.

– The dissociation process is much simpler. Here, the rate of decay which you see is equal to the off rate. Just dissociation is happening, and the dissociated analyte is carried away from the surface by the flow.

5

Kinetics 101

� Definitions of the rate constants:

� Association rate constant, ka:

– The rate of complex formation, i.e. the number of AB complexes formed per second in a 1 molar solution of A and B

– It has the units M-1s-1, and typical values are from 103 to 107 M-1s-1

� Dissociation rate constant, kd:

– Stability of the complex, i.e. the fraction of complexes that decays per second

– kd= 0.01 s-1= 1% of the complexes decays per second

– So, the larger the number, the faster the dissociation process

– It has the units s-1, and typical values 10-2 to 10-5 s-1.

6

Kinetics 101

� Rate and Equilibrium constants are related:

• Association rate:

• Dissociation rate:

• At equilibrium: Association = Dissociation

• The equilibrium constant:

[ ][ ] [ ]BAk

dt

ABda ⋅⋅=

[ ][ ]ABk

dt

ABdd ⋅=−

[ ] [ ] [ ]ABkBAk da ⋅=⋅⋅

[ ]

[ ] [ ] d

aA

k

k

BA

ABK =

⋅=

[ ] [ ]

[ ] a

dD

k

k

AB

BAK =

⋅=

A + B ABka

dk

7

Importance of Assay Development and Optimization

8

Things To Consider Before Setting Up a BLI Experiment

• Hydrate biosensors in assay buffer for at least 10 minutes

• Allow all reagents to get to room temperature

• Use only Greiner black plates

• Size of ligand and analyte

• Building up the layers from

• Affinity of the interaction – anything known about expected affinity?

• 0.1 KD KD 10-20X KD

• Stoichiometry of binding – 1:1, 2:1, 1:2 etc…

• Quality of ligand and analyte – percent activity, purity, before and after modification and binding to the biosensor

• Quality of reagents is a key to a successful assay!!

9

Pick an Appropriate Assay Format

Capture-Based Immobilization Direct Immobilization

Benefits

• No assay development for covalent bond

• Compatible with crude samples

• More homogenous surface with capture

epitope specificity and known point of

immobilization

Drawbacks

• Capture of ligand may not be with highestaffinity

ONH NH

= ligand; protein immobilized

= analyte; target protein

Benefits

• Very stable binding

• Compatible with most proteins

Drawbacks

• Requires purified protein

• May create a heterogeneous surface

10

Ligand Loading Optimization Must Be Done

11

Optimization of Ligand Loading Density

12

Ligand Immobilization

13

Analyte Concentration Series

• Dissolve analyte in best buffer for solubilization

• Using optimal ligand loaded biosensor, perform titration series of analytefrom 10x KD to 0.1x KD

• Include buffer only ( Zero analyte ) control to check for buffer drift

• Include no ligand control to check for non specific binding

• Allow enough dipping time in association step to obtain signal saturation behavior for at least the highest concentration of analyte

• Allow enough dipping time in dissociation step to obtain at least 5% dissociation of signal for the highest analyte concentration

• Use the same assay buffer for baseline-association-dissociation steps (avoid buffer mis-match and signal variations resulting from refractive index changes from the buffers)

14

Analyte Association

Time

Response

Slower phase never reaching equilibrium

Incomplete dissociation

1:1 Binding

?

15

Analyte Association

Time

Response

Slower phase never reaching equilibrium

Incomplete dissociation

1:1 Binding

16

Heterogeneous Binding

• Heterogeneous binding � NOT THE END OF THE WORLD

• Heterogeneous binding signal is typically the result of a non-specific binding interaction contaminating the specific binding signal

• Take steps to eliminate the non-specific interaction

• Analyte concentrations may be too high

• Ligand loading level may be too high

• Flipping assay orientation

• Association and dissociation times long enough to get accurate on rates and off rates

• Different biosensors types to get best 1:1 binding between ligand and analyte optimized

• Assay buffer may need some optimization to reduce non-specific interactions

• Reference sensor may show similar non-specific signal increase, which may be subtracted from the binding signals

17

Minizing Non-specific binding in an Assay

18

Regenerate

Sensor back to

original surface

Capture human IgG or

human Fc containing ligand

Regenerate

Capture molecule covalently

immobilized or biotinylated

Analyte

removed

Anti-Human

IgG Capture

Amine

Reactive

Or

Streptavidin

New sample

New hIgG capture molecule

Analyze

kinetics

Analyze

kinetics

Optional Regeneration of Biosensors

Regenerate

Analyte

removed

Quantitate

analyte

Protein A

New sample

19

Biosensor Regeneration

20

Successful Biosensor Regeneration

21

Buffer

Capture molecule

Analyte of Interest Bin

din

g (

nm

)

Time

BaselineLoading

Baseline

Association Dissociation

• 8,16, 32, 48 or 96 samples can be analyzed in parallel

• Measure on rates and off rates

• Data is displayed in real-time

• Experimental protocols can be customized

Octet Biosensors

Octet Automated Workflow for Kinetics

22

Analysis of Results – What is good data?

After curve fitting has been performed – it is necessary to evaluate the quality of the fit and the reliability of the calculated binding and affinity constants.

• Visually inspect the data and see if the fit lines (red) conform well to the data traces if the fit lines are far from actual data fit is not ideal

• If highest and lowest concentrations do not behave as rest then you can exclude can improve fitting in global analysis

• Is the KD consistent with expectations on literature or previous knowledge of interaction

• Residual values should not be + 10% of the maximum response of the fitted curve

• Error values are provided in the analysis table for Ka and Kd acceptable within one order of magnitude of the rate constant values

• R2 values indicate how well the fit and experimental data correlate and above 0.95 are considered a good fit

• X2 is the sum of the squared deviation should be generally below 3. X2 is the measure of error between the experimental data and the fitted line. The smaller the X2 indicates a better fit

23

Setting up Kinetics Assays

• Use experiment templates for optimization

assays such as pH scouting, regeneration

scouting

24

Setting up Kinetics Methods

• Enter analyte concentrations in MOLAR terms and include at

least 1 well of buffer (0nM Sample) for referencing

25

Setting up Kinetics Assays

•Set Shake speed to 1000rpm.•Remember: the analysis software needs Baseline, Association and Dissociation steps consecutively to work

26

Setting up Kinetics Assays

� If the KD is in the nM range, an association of 10 minutes and a dissociation of 10-20 minutes may be sufficient to obtain kinetic constants with low error.

� If the KD is < 1 nM, an association of 15 minutes and a dissociation of 30-60 minutes may be necessary to obtain kinetic constants with low error.

� If the KD is unknown, err on the long side for both association and dissociation, skip ahead in acquisition software, and/or shorten data analysis times in the software.

• Use the ‘go to next’ and ‘extend’ step buttons at run time to optimize timings during assay development. Do not do this if you intend to run double referencing!

27

Setting up Small Molecule Kinetics Assays

Use experiment templates then modify as required

For low affinity samples, set up the plate with dilution series running across, one

sample per sensor:

‘Row Format’

28

Setting up Small Molecule Assays

For high affinity samples, set up the plate with dilution series running down, one sample per 8 sensors:

‘Column Format’

Use “slow dissociation” experiment template

(column format) and modify as required

For High Affinity Interactions: when koff < 10-3

29

Setting up Small Molecule Assays

Set up Target Sensors and reference sensors (all Super Streptavidin, S

Ideally, reference sensors will have a similar, but inactive target immobiliseIf this is not possible, they should be blocked with, for example, biocytin

30

Kinetics Analysis Considerations

• Processing parameter recommendations:

• Y-axis alignment (last 5-10 seconds)

• When to use interstep correction (baseline/dissoc same well, avoid with fast kinetics)

• How to select analysis model and use steady state

• How choose Rmax linked or unlinked (row vs column assay format)

• What to look for in analysis do determine good fits (visual inspection, R2, X2)

31

Kinetics Data Analysis

Subtract reference well/sensors

Align Y axis to baseline, change time range

Process data then open ‘Analysis’ tab

Interstepcorrection if baseline/dissociation in same wells

Define reference wells/sensors

32

Kinetics Data Analysis

First, Change the ‘well type’ for the zero concentration sample from ‘sample well’ to ‘reference well’

33

Kinetics Small Molecule Analysis

During data analysis, use the ‘Double Referencing’ option if appropriate

Other options should be the same as for Protein / Protein Assays

34

Kinetics Data AnalysisCheck steps for analysis

Select model

Select fit type (local/global). If by ‘Group’, group by Color

Fit Curves

Select appropriate Rmaxsetting

Adjust step times if necessary

Use steady state if

appropriate and only if all

curves reach equilibrium

35

Best Practices for Octet Systems

36

Important that correct plates are used with correct volumes

� A minimum of 2 microplates is required for every Octet assay (pre-wet plate and sample plate)

� HTX, QK 384 and RED 384 models can run either 96 or 384-well plates

� The Octet QK, Qke and RED96 use regular 96-well black plates are 200ul volume must be used for RED96. Greiner catalog number 655209

� Half area black 96 well plates can be used with QK / QKe / QK384/ RED384/ HTX Greiner catalog number 675076

� Full volume black 384-well plates require 120 uL Greiner catalog number 781209.

� Tilted-well black 384 well plates recommended volume is 40 uL – 80 uLavailable only though ForteBio (catalog number 18-5080 (Pack of 10), 18-5076 (Case of 100)

� Sample is recoverable and measurement non-destructive.

37

General essentials for all assays

�Keep instrument lamp on for best life and performance

�Always pre-wet sensors for 10 minutes.

�Use the same matrix / buffer as the samples for prewettingstep.

�Maintenance commitment is low. Clean the sensor ‘pickers’ regularly with 70% ethanol to avoid plastic build up from the sensors. If this is ignored, problems may bee seen with irregular pick-up of the sensors.

38

Thank you for your attentionAny questions?