Affinity Measurement with B iomolecular I nteraction A nalysis Bia core

1. Affinity Measurement withBiomolecular Interaction AnalysisBiacore

2. Kinetics Affinity Thermodynamics What SPR Biosensors Measures Concentration Specificity How fast, strong & why… Is the binding of a lead compound How specific & selective... Is this drug binding to its receptor? How much... Biologically active compound is in a production batch?

3. Biacore History • Founded 1984 as Pharmacia Biosensor AB • Biacore System launched October 1990 • Biacore Symposium 1991 • Inline referencing started 1994 • Became Biacore AB in 1996 • Support of regulated environments from 2002 • Entering the drug discovery market with S51 in 2002 • Going into protein arrays with Biacore A100 and Flexchip in 2005

4. Probing Biological Affinities

5. SPR Detection Sensor Chips IFC Microfluidic The Corner-stones of the Technology

6. The SPR Detector

7. Total Internal Reflection & SPR • Gold layer • Evanescent field • Total Internal Reflected light (TIR) • TIR angle • Incident Light • High refractive index medium: Prism • Low refractive index medium: Buffer

8. Principle SPR detects refractive index changes close to the surface E.g. accumulation of 1 pg/mm2 gives a change of 1 µRIU or 1 RU All biomolecules have refractive properties, so no labeling required Result No need to separate bound from free This facilitates real-time measurements as a basis for taking kinetic data Work with un-altered analytes possible SPR detection

9. Sensor Chips Sensor Chip specific matrix Glass Gold 50 nm

10. Sensor Chip CM5 • Dextran matrix covered with carboxyl groupes (red circles) • Captures ligands such as proteins, lipids, carbohydrates and nucleic acids(irreversible) • Study of analytes ranging in size from small organic molecules, e.g. drug candidates, to large molecular assemblies or whole viruses.

11. Sensor Chip CM4 • Similar to CM5 but with a lower degree of carboxymethylation resulting in low immobilization capacity and lower surface charge density. • Allows to reduce non specific binding in case of complex mixture such as cell extract or culture medium. • Advantageous for kinetic experiments where low immobilization levels are recommended.

12. Sensor Chip CM3 • Similar to CM5 but with shorter dextran chains, giving a lower immobilization capacity of the surface. • Allows the interaction to take place closer to the cell surface which can improve sensitivity when working with large molecules, molecular complexes, viruses or whole cells.

13. Sensor Chip SA • CM dextran matrix pre-immobilized with streptavidin • Captures biotinylated ligands such as carbohydrates, peptides, proteins and DNA (irreversible) • Ideal for capture of large biotinylated DNA fragments and study of nucleic acid interactions

14. Sensor Chip NTA • CM dextran matrix pre-immobilized with nitrilotriacetic acid (NTA) • Capture of His-tagged ligands via metal chelation • Controled steric orientation of ligand for optimal site exposure • Regeneration by injection of EDTA to remove metal ions

15. Sensor Chip L1 • CM dextran matrix modified with lipophilic anchor molecules • For rapid and reproducible capture of lipid membrane vesicles such as liposomes,with retention of lipid bilayer structure • Allows studies of transmembrane receptors in a membrane-like environment , for example.

16. The Steps in the Biacore Assay Surface preparation Analysis Cycle

17. a n a l y t e l i g a n d a n a l y t e l i g a n d c a p t u r i n g m o l e c u l e Surface Preparation: Immobilization Direct Capture

18. Direct Immobilization

19. Various Coupling Chemistries

20. Amine Coupling - Sensorgram • Activation = EDC/NHS injection  surface esters • Ligand contact = reaction with amine groups on ligand • Blocking = deactivation of free esters with ethanolamine Blocking Activation Ligand contact

21. High Affinity Capture

22. Capture Surfaces and Molecules

23. Analysis Cycle Generates the desired data Sample injection Regeneration Evaluation

24. Analysis Cycle • Done by • Buffer flow, pH shift, salt & chaotrophic ions, detergents • Similar concept as in affinity chromatography • Results • Re-use of biospecific surface • Low amount of ligand needed Sample injection Regeneration Evaluation

25. The Result: the Sensorgram

26. Experiments without Kinetics Specificity Multi layer structure Concentration assays Affinity constants

27. Specificity • Do two molecules interact with each other? Yes/No Answers. • Different analytes are tested with the same ligand e.g. different lectins with immobilized thyroglobulin. • Quantitative measurements, test a range of analyte concentration to determine the concentration dependency of the response.

28. Specificity Analysis Overplay plot of sensorgrams showing interaction between different lectins and immobilized thyroglobulin.

29. Multiple Binding • Enhancement • Enhancing lower detection limit of assays • Sandwich assays • Enhancing selectivity of test • Epitope mapping • Charting the surface of antigens with antibodies • Multimolecular complexes • Identify the logical sequence of binding events

30. Analyte Ligand 2nd Binder 31000 30000 29000 28000 27000 26000 50 100 150 200 250 300 350 400 Multiple Binding Response [RU] Time [s]

31. Epitope Specificity of two mAbs against HIV1-p24 Immobilization of rabbit rabbit anti-mouse IgG1 • A: baseline • A-B: 1st mAb against HIV1-p24 • B-C: blocking antibody • C-D: HIV1-p24 • D-E: 2nd mAb against HIV1-p24

32. Concentration Assays • Concentration based on biological activity • All concentration assays require a calibration curve • Concentrations of unknowns samples are calculated from this • 4 - 7 concentrations in duplicate • Calibrants and unknowns in same matrix • Moderate to high densities on sensor chip • Direct binding formats • Inhibition formats

33. Sample Calibration Curves Response x x x x x Concentration = Sample matrix for calibration curve Sample matrix for unknown samples

34. Affinity Analysis • How STRONG is the binding at equilibrium? • » Quantify KD • » Rank Antibodies • » Find best Ab pairs

35. Signal [RU] 20 15 10 5 0 Time [s] 60 120 0 Affinity and Equilibrium • Furosemide binding to carbonic anhydrase • Referenced data • Report Point towards end of injection • Do secondary plot

36. Determining Affinity Constants • Plot Req against C • Steady state model • Concentration at 50% saturation is KD

37. How FAST is the binding ?» ka kon (recognition)» kd koff (stability)» KD = kd/ka» Ab selection; wash steps Kinetic Analysis

38. All target sites occupied 1 µM 100 nM 30 min 60 min 30 min 60 min Same Affinity but different Kinetics • All four compounds have the same affinity KD = 10 nM = 10-8M • The same affinity can be the result from different kinetics ka [M-1s-1] kd [s-1] 106 10-2 105 10-3 104 10-4 103 10-5 KD 10 nM

39. Rate Constants

40. kd (A).(B) = ka (AB) Equilibrium Constants ka (AB) = kd (A).(B)

41. Equilibrium and Kinetic Constants are related

42. Equilibrium and Kinetics in Biacore

43. Information in a Sensorgram

44. dR = ka. C . (Rmax-R) – kd . R dt Extracting Rate Constants from Sensograms • Measure binding curves • Decide on a model to describe the interaction • Fit the curve to a mathematical rate equation describing the model • e.g. • Obtain values for the constants ka, kd, Rmax • Assess the fit • overlay pots, residual plots • acceptable statistics e.g. chi2 – curve fidelity • Biological and experimental relevance of the calculated parameters

45. Biacore and other Methods Conventional Biacore Assays Time Method Time One Day ELISA Day 1 Isotyping RIA Weeks + labelling Affinity Day 1&2 Na Na Kinetics Day 1&2 Weeks + labelling ELISA Overnight Epitope Map Biacore is much quicker than conventional methods

46. Summary Surface plasmon resonance detects binding events as changes in mass at the chip surface Real-time kinetic measurements Qualitative rankings Measurement of concentrations Information about structure-activity relationships No labeling and low volumes samples needed