Reichert - Lunch & Learn Presentation

Reichert Life Science Legacy

• 15 years of SPR expertise

• >200 publications on Reichert SPR

• Cover the full spectrum of bio molecular interactions – Protein-protein – Protein-small molecule

• Ensuring success through service– SPR support staff helps researchers solve problems– Methods development, high-volume experiments, feasibility studies

A history of exceptional performance, value and support.

The Reichert SPR Advantage • Your partner every step of the way

– Unmatched customer service and support solutions– Maximum uptime drives better results

• Solve your research bottlenecks– Scalable to research and lab needs– Systems accessible to your lab

• Reliable binding, kinetics, concentration and thermodynamic data

– Helping you answer questions quantitatively

• Increase your sample flexibility– Broader application options– Robust fluidics

• Reduce your equipment and maintenance costs— Lower operating, lifetime ownership costs

Better Fluidics, Better Results

• Run sample types you wouldn’t consider on other systems• Minimize maintenance requirements and costs

– Five-year total cost of ownership is 30% lower than leading competitor

– Tubing is easy-to-access and replace

• Variable tubing sizes – From 64 to 500 m inner diameter

• Variable sample loop volumes – From 10 to 5000 L inner diameter

• Compatible with a wide range of solvents

Advantages Over Biacore

• Robust, easy-to-access fluidics system• Specialized flow cells • User-friendly software interface• Lower capital investment and lifetime

cost of ownership • Superior, on-demand support services• Unmatched baseline stability• Comparable sensitivity and noise performance

Advanced SPR Technology

• Innovative two and four channel systems– Ideal sensitivity for low molecular weight analysis

• Noise and sensitivity performance required for challenging applications

• More applications and sample types– Robust enough for crude samples,

cell lysates, aggregates

• High sample capacity – Two 96- or 384-well plates– Up to 768 samples—or any combination

of plates and vials

• Scalable to meet your needs now and later– Solutions grow as you do – Rental services available

Commitment to Service and Your Success

• Application and methods development • On-call assistance to minimize downtime• In-lab contract sample analysis for customers• Installation training, lectures and hands-on

application support• Preventative maintenance• Remote and on-site service plans• Additional consulting services

Current Customer Sampling

Reichert4SPR System • New in 2015• Increased throughput for drug discovery

– 4 channels improve the efficiency and study design flexibility

• Advanced optics, image sensing, electronics and software

• The most robust fluid handling systems– Accommodates many more sample compositions

• Reliable results and seamless data analysis• Quick method development and

programmability• Far easier to maintain than other SPR instruments

– Max uptime to drive better results

SPECIFICATIONS

Reichert4SPR Key Specs

Reichert 7500DC Specs

CompetitiveComparisons

Reichert4SPR Biacore T200

Reichert4SPR vs GE-Biacore T200

• Equivalent performance

• Reichert Advantages:

• Robust Fluidics

• Better uptime• More flexible• Affordable

Reichert4SPR Biacore T200

Reichert 2 Channel SPR vs GE-Biacore 2 Channel SPRReichert 7500DC Biacore X100

Channels 2 2Injection of Lysates, Cells, Particulates

Yes No

DIY Fluidics Yes No

Sample Capacity 768 samples 15 SamplesBaseline Noise 0.05µRIU RMS <0.3µRIU RMSBaseline Drift 0.01µRIU RMS <0.1µRIU RMS

Sensor Chip Cost $84 dextran chip $189 dextran chipSpecialized Flowcells Electrochemistry, Photochemistry,

Flourescence, MALDI MS plugnone

Sample Volume 1-4500µL 20-30µL

Temperature Range 10oC below ambient to 70oC 10oC below ambient to 45oCSample Compartment Temperature

4oC or Ambient None

Flow Rate Range 0.1-3000µL/min 1-100µL/minMin Molecular Weight <100 Da 100 Da

SENSOR CHIPS

Reichert Sensor Surfaces (I)

• Low Binding Capacity• No Matrix Depth• Large/Moderate MW

Molecules• Low Non-Specific

Binding

• High Binding Capacity• Flexible• Large/Moderate or Low

MW Molecules• Minimal Non-Specific

Binding

= COOHCarboxyl Surfaces

Reichert Sensor Surfaces (II)

• Steptavidin Amine Coupled• Capture Biotinylated Ligands• Stable Surface

• Alkyl Surface• Capture Lipid Monolayer• Regenerate with CHAPS

• Nitriloacetic Acetic Acid Surface• Capture HIS-Tagged Ligands• Decaying Surface• Regenerate with Imidazole

• Protein A Amine Coupled• Capture IgGs• Regenerate with pH 2 buffer

SPR TECHNOLOGY

Surface Plasmon Resonance

Metal SurfacePlasmons (Electron Waves)

Surface Plasmon Resonance AngleIn

tens

ityDetecto

r

780

nm L

ight

> qc Resonance (Energy Transfer)

Surface Plasmon Resonance

Angle

Inte

nsity

Change in Refractive Index

Changes in Refractive Index

Angle

Inte

nsity

Mass Sensor

Detecto

r

Angle

Inte

nsity

Time

Res

pons

e

Raw Data

Response Data

780

nm L

ight

Response Units

Time

Res

pons

e

Units are in RIU (10-6 refractive index units)

1 RIU = 1 pg/mm2 of mass binding

A very precise refractometer

A very precise mass sensor

Versatile Technique

Time

Res

pons

e

Binding Reponse

Baseline

Is there an interaction? (Yes/No Binding)

How strong is the interaction? (Affinity)

How quickly do they interact and dissociate? (Kinetics)

Why? (Thermodynamics) (DH, DS, DG)

How much? (Concentration)

Asso

ciatio

nDissociation

Regeneration

All Classes of Biomolecules

<100 Da to Proteins to Cells

• Proteins• Lipids• Carbohydrates• Nucleic Acids

• LMW Molecules

• Whole Cells • Bacteria, Viruses

The Importance of Kinetics

Series1

0.0001 0.001 0.01 0.1 1102

103

104

105

106

107

kd (s-1)

k a (M

-1s-1

)

10 pM 100 pM 1 nM 10 nM

1 M

10 M

100 M

1 mM

100 nM

KD

KD(M) = kd (s-1)ka (M-1s-1)

Conventional Regeneration Approach

SampleReference

17900

18100

18300

18500

18700

18900

1000 2000 3000 4000 5000 6000

Time (sec)

Res

pons

e (

RIU

)

• Each concentration in a single cycle• Requires regeneration between

injections

Equilibrium Data

KD = 940 nM

Measuring Responses at Equilibrium

Steady-State Measurement - not Kinetics

Another way to measure KD

aside from kinetics

Thermodynamics

0

20

40

60

80

0 50 100 150 200 250

Time (sec)

Resp

onse

(uRI

U)

Time (sec)

Res

pons

e (u

RIU

)

20 oC

35 oC

Measure Binding at Different Temperatures (at least 5)

Van’t Hoff Equation

Van’t Hoff Plot

Slope = DHR

-14.4

-13.8

-13.2

-12.63.2 3.3 3.4 3.5

1/T (X 10-3 K-1)

ln (K

D)

Y-Intercept = DSR

Concentration Determination

Determine Concentration of Biomolecule in Solution

Surface with Specific Antibody

APPLICATIONS

•Anti-HSA IgG / HSA•Enzyme-Inhibitor Interactions•Lipid-Peptide Interactions•Carbohydrate-Receptor Interactions•Development of a Cytokine Antagonist•Cell-Protein Binding

APPLICATION 1: ANTI-HSA IGG/HSA

Ligand: Anti-HSA IgGAnalyte: Human Serum Albumin (HSA)

Amine Coupling

animation

animation

Anti-HSA Immobilization

10000

15000

20000

25000

30000

35000

0 500 1000 1500 2000 2500 3000 3500 4000 4500

Time (sec)

Res

pons

e (u

RIU

) EDC/NHS

Anti-HSA

1M Ethanolamine, pH 8.5

HSA Injections

Sample

Reference

17900

18100

18300

18500

18700

18900

1000 2000 3000 4000 5000 6000

Time (sec)

Res

pons

e (

RIU

)

HSA Kinetic Results

APPLICATION 2: ENZYME-INHIBITOR INTERACTIONS

Ligand: Carbonic Anhydrase IIAnalytes: 4-Carboxybenzene Sulfonamide (200 Da) and Methane Sulfonamide (95 Da)

Inhibitor Kinetics4-CBS

Methane Sulfonamide

KD = 857 nM

KD = 650 M

APPLICATION 3: LIPID-PEPTIDE INTERACTIONS

Hydrophobic Sensor ChipPrepare Liposome SurfaceInject Peptide

Vesicle CapturePS Vesicles Capture on Phytosphingosine Surface

10000

10500

11000

11500

12000

12500

13000

13500

14000

14500

15000

10550 11550 12550 13550 14550 15550

Time (sec)R

espo

nse

(uR

IU)

Sample ChannelReference Channel

3260 uRIU of PS Vesicles on surface

• Lipophilic surface preserves vesicle bilayer conformation

• QCM dissipation data verifies intact vesicles

Phosphorylated Protein

KD = 3.92 nM

KD = 467 pM

APPLICATION 4: CARBOHYDRATE-RECEPTOR INTERACTIONS

Carbohydrate-Receptor Interactions

• Direct SPR assay to investigate carbohydrate-lectin binding kinetics

• Concanavalin A (Con A) and mannose derivatives used as a model

• Gain insight into the fundamental mechanisms of multivalent carbohydrate binding

• Methyl-a-D-mannopyranoside used as a control

• Three clicked mannosylated GATC dendrimers

Eva Maria Munoz; Juan Correa; Eduardo Fernandez-Megia; Ricardo Riguera. JACS 2009, 131, 17765-17767.

Sensorgrams

• Monosaccharide/Con A binding is comprised of fast on and off rates that follow a 1:1 binding model

• Glyco-dendrimers have more complex binding kinetics consistent with the multivalent nature of the dendrimers and the clustered arrangement of lectin

• Association rates are similar but dissociation rates varied with the generation of dendrimer


Binding Mechanism Unraveled

• Three-phase binding model proposed:1) A low affinity binding site similar to the monosaccharide2) A higher affinity binding mode resulting from dendrimer binding with higher functional valency3) As dissociation occurs from the low affinity binding site, the sites become occupied by higher

functional valency dendrimers.


APPLICATION 5: DEVELOPMENT OF A CYTOKINE ANTAGONIST

Design of the Antagonist

• IL-1 Cytokines are master mediators of the inflammatory response.

• IL-1α and IL-1β protein are elevated in the lacrimal gland, tears, and the ocular surface in all forms of dry-eye disease

• Chimerized two IL-1 receptor ligands, IL-1β and IL-1Ra, to create an optimized receptor antagonist.

Jinzhao Hou, et.al. PNAS 2013, 110, 3913-3918.


SPR Sensorgrams

SPR Results

• Focused on chimera 93:60


APPLICATION 6: CELL-PROTEIN BINDING

Human Embryonic Kidney

HEK Cells Capture

200 nM Fibrinogen Injection

Surface Heterogeneity Model

ka1 = 2.40 e3 M-1s-1

kd1 = 8.51 e-3 s-1

KD1 = 3.54 M

ka2 = 9.74 e3 M-1s-1

kd2 = 2.57 e-4 s-1

KD2 = 26.4 nM

SPECIALTY APPLICATIONS

•Electrochemistry

•Mass Spectrometery

•Photochemistry/Fluorescence

Documents

Reichert - Lunch & Learn Presentation