WilBio 07

Providing Technologies to Accelerate Therapeutics Development

. Confidential Information

A Label-Free, Real-Time Detection Systemfor Molecular Interaction Analysis

. Confidential Information

The Octet InstrumentEnabling rapid sample analysis

. Confidential Information

Two-dimensional binding surface

Biocompatible Matrix- minimizes non-specific binding

Uniform

Non-denaturing

BLI Surface ChemistryBio-Layer Interferometry

|---- 600 μm ----|

. Confidential Information

A layer of molecules attached to the tip of an optic fiber creates an interference pattern at the detector.

BioLayer Interferometry (BLI)Proprietary new technology for label-free detection

. Confidential Information

A layer of molecules attached to the tip of an optic fiber creates an interference pattern at the detector.Any change in the number of molecules bound causes a measured shift in the pattern

BioLayer Interferometry (BLI)Proprietary new technology for label-free detection

WilBio 07

Rapid Quantitation of Antibodies from bioreactors using the Octet System

. Confidential Information

Anti-hIgG Fc sensoror

Protein A sensor

IgG standard Purified sample,Cell culture supernatant,

Crude lysate,Etc.

Biosensor tip

Capture molecule

standard

Unbound molecule

unknowns

Octet QuantitationAutomated Workflow

. Confidential Information

Octet Automated Workflow for QuantitationProtein A Biosensors

Standards

TestSamples

Bin

ding

(nm

)

Time (sec)

Bin

ding

Rat

e

Concentration

Data is taken for 2 minutes per 8 wells 96 wells in ~30 minutes1 step, no washing

Octet Biosensors

120

. Confidential Information

Octet Binding Curve = rate of increase in optical thickness as the sample binds to the sensor.Different protein concentrations result in different binding curves

Quantitation – Real-time binding curvesRate of binding correlates to concentration

Rate of binding is proportional to concentration

700 ug/mL500 ug/mL300 ug/mL

100 ug/mL

30 ug/mL

10 ug/mL

3 ug/mL

1 ug/mL

. Confidential Information

Accurate quantitation is derived from the standard curve

Accurate Quantitation MeasurementRate of Binding used to plot standard curve

N=8AVE calc.

conc.%CV conc.

700 700.0000 9.1%500 504.0032 6.6%300 302.4974 4.8%100 100.2989 2.4%

30 30.0245 2.0%10 9.9845 1.7%

3 3.0017 3.5%1 1.0005 4.3%

. Confidential Information

The Octet SoftwareQuantitation Analysis and Reporting

Automatic analysis following data collectionAlerts to flag standards that fall outside expected rangeStandard curve exportMicrosoft Word and Excel format Quantitation Reports

. Confidential Information

>10% CVELISA

< 5% CVHPLC

< 10% CVOCTET

PrecisionPer Plate

Octet throughput and reproducibilityWorkflow and precision compared to ELISA and HPLC

. Confidential Information

25 μg/mlN = 6 instrumentsN = 6 sensor lots

ID OCTET #2

OCTET #4

OCTET #5

OCTET #6

OCTET #7

OCTET #9

Octet Mean

% CV 3.6% 5.0% 6.7% 4.2% 6.0% 7.2% 7.5% N 24 24 24 24 24 24 192

Instrument and sensor reproducibilityGood correlation between instruments and sensor lots

. Confidential Information

. Confidential Information

. Confidential Information

130 µg/ml132 µg/ml100 µg/ml

327 µg/ml323 µg/ml333 µg/ml

47 µg/ml42 µg/ml31 µg/ml

281 µg/ml280 µg/ml248 µg/ml

0 µg/ml0 µg/ml0 µg/ml

OctetCrude

OctetCentrifuged

HPLCCentrifuged

Only Octet provides data from crude samplesData correlates well with cleared lysates

Octet Quantitation with crude samplesIgG Expressed in E.Coli analysed by OCTET and HPLC

. Confidential Information

Correlates well with HPLCBroad matrix compatibilityMinimal interference from crude lysatesHigher throughput than HPLC and manual ELISA

Octet – Quantitation AnalysisSummary

WilBio 07

Kinetic Characterization of Proteins in Real-time using the Octet System

. Confidential Information

Protein 1 + Protein 2 Complexka

kd

kobs – kd[Conc (M)]

ka= KD = kdka

ka = rate of association or “on-rate”kd = rate of disassociation or “off-rate”

Binding Kinetics: The Basics

. Confidential Information

Octet Automated Workflow for KineticsStreptavidin Biosensor

BufferLigand-BiotinBufferProtein of interestBuffer

Bin

ding

(nm

)

Time

Baseline Loading Baseline Association Dissociation

8 samples can be analyzed in parallelData is displayed in real-timeExperimental protocols can be customized during sample programming

Octet Biosensors

. Confidential Information

Octet Automated Workflow for Kinetics

. Confidential Information

The Octet SoftwareKinetics Analysis and Reporting

User software enabling graph exporting in a number of different formatsStreamlined data analysis and model fitting within Origin 7.5Microsoft Word format Kinetics Reports showing curve fitting, residuals,ka, kd and KD

0 1000 2000 3000 4000 5000

0.0

0.2

0.4

0.6

time (sec)

nm

Global FitModel 1:1

0 1000 2000 3000 4000 5000-0.10

-0.05

0.00

0.05

0.10

time (sec)

. Confidential Information

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000

Activation(EDC/NHS)

Immobilization(receptor F)

Quench(EtAmine)

Binding(ligandscreening)

Dissociation

Immobilization CV 4.8%

Customer Data from Customer G: Screening of ligands for Receptor F using Amine Reactive Biosensors

nm

Time (sec)

. Confidential Information

Clear Results from a Ligand Screen

kdis

s

kassoc

. Confidential Information

“What was impressive for us was the ability to use the media and crude peri samples to predict the affinities of the pure samples. The overall ranking was very clear and the machine has been extremely easy to use.”

- Pete FlynnDirector, Biochemistry Research, KaloBios

Broad sample typesoff-rates from periplasmic fractions vs. purified Fabs

Data provided by KaloBios

. Confidential Information

Correlation of Kd from Sonata and Biacore

R2 = 0.9554

0.00E+001.00E-082.00E-083.00E-084.00E-085.00E-086.00E-087.00E-088.00E-089.00E-081.00E-07

0.00E+00 2.00E-08 4.00E-08 6.00E-08 8.00E-08 1.00E-07Kd Sonata (M)

KD

Bia

core

(M)

Data analysis performed for both systems assuming pseudo 1st order kinetics.

KD Octet (M)

Correlation of KD from Octet and Biacore

Octet data correlates well with Biacore

KD

KD

. Confidential Information

Flexible Assay FormatsMeeting a variety of assay and throughput needs

. Confidential Information

Simple workflow

Rapid assay development

Correlates well with Biacore resultsEasy data analysis

Reliable and reproducible kinetic reporting

Compatible with crude samplesFlexible formats for high throughput

Higher throughput for rank ordering of clones

Long off-rates for kinetic determination

Octet – Kinetics AnalysisSummary

. Confidential Information

Applications in Bioprocessing Development

Quantitation Applications- IgG quantitation- Protein quantitation

Cell line developmentBioreactor process optimization Production titer monitoring

Kinetics Applications- Affinity characterization- Measure kinetic constants- Rank order affinities

Rank Ordering of clonesClone SelectionAntibody characterizationLigand Screening

. Confidential Information

AcknowledgementsFortéBio R&D team

KaloBiosPete Flynn

Raven biotechnologyTri DoTim HotalingJustin GrahekLaura Lerner

Visit us at booth # 26

Come see our posters:

Label-free Screening and Selection of Multiple Antibody-Antigen Pairs on the Octet System

Streamlining Assay Development with Amine Reactive Biosensors on the Octet System

. Confidential Information