BIAcore: Real-Time Biomolecule Interaction Analysis

Transcription

1 BIAcore: Real-Time Biomolecule Interaction Analysis Biacore T-100 奇異亞洲醫療設備股份有限公司林怡欣產品專員 Understanding molecular recognition a fundamental phenomenon of life How strong => Affinity How fast => Kinetics Why that strong => Equilibrium thermodynamics Why that fast => Transition state thermodynamics Important know-how for structure-based molecular design: Drugs and Engineered antibodies 14 / GE Biacore T100 system presentation / 3/28/2008 1/

2 Biacore T100 Biacore T100 Introduction Hardware features Software overview Control Software Evaluation Software New Consumables Sensor chips Buffers Biacore T100 GxP Package 17 / GE Biacore T100 system presentation / 3/28/2008 2/

3 Biacore T100 system features Based on S51 platform Novel fluidic system Buffer selector valve Integrated buffer degasser Integrated sample cooling Sample capacity 384 micro plates 19 / GE Biacore T100 system presentation / 3/28/2008 Fluidic system - Optimized for highest quality kinetics 4 flow cells single, pair wise and serial run Measure the fastest on rates to the slowest off rates Pair-wise on-chip flow cell connections give good reference subtraction The flow cells are optimized for use in pairs (FC1+FC2, FC3+FC4) IN Measure off rates as fast as 0.5 s -1 FC 1 Many IFC functions inherited FC 2 OUT Open valve from BC 3000 FC 3 Closed valve Dual inject (Co-inject), mix FC 4 20 / GE Biacore T100 system presentation / 3/28/2008 3/

4 Optimized for highest quality kinetics Measure the fastest on rates to the slowest off rates Pair-wise on-chip flow cell connections for good reference subtraction 4 flow cells - single, pair wise and serial runs Measure off rates as fast as 0.5 s-1 RU Time Response RU Time 21 / GE Biacore T100 system presentation / 3/28/2008 Buffer selector Compare buffer effects in one single run Assay development - Scout for optimal assay conditions Study environmental effects (e.g. ph, salt) on binding properties Both mechanistic and stability studies Controlled from the software Buffers can be changed when running buffer scouting wizard or during method runs Up to 4 different buffers can be used Κ (νμ) ph of assay buffer affinity 3 Fabs showing a similar pattern of ph effects - marked increase in affinity at ph very minor change at ph / GE Biacore T100 system presentation / 3/28/2008 4/

5 Compare buffer effects in one single run Study environmental effects on binding properties Both mechanistic and stability studies Use buffer scouting for fast assay development Up to 4 different buffers can be used 3 Fabs show a similar pattern of ph effects marked increase in affinity at ph < 7.5 very minor change at ph > / GE Biacore T100 system presentation / 3/28/2008 Study interactions at physiological temperatures Integrated buffer degasser Improves robustness when studying interactions at elevated temperatures Analysis temperatures range 4 45 o C No need to de-gas buffers Ηιγη Response RU Extremely stable and reproducable data at 37 degrees! Time s 24 / GE Biacore T100 system presentation / 3/28/2008 5/

6 Liquid handling system IFC To waste Buffer degasser Syringe pumps Needle S2 S1 Vacuum degasser Peristaltic pump Buffer selector valve Buffer selector valve A B C D Buffer Waste Water 25 / GE Biacore T100 system presentation / 3/28/2008 Capacity - Run up to 384 samples per run Supports 96 and 384 well microplates 48 hours unattended run time Integrated sample cooling for temperature sensitive samples From 4 ºC to 45 ºC (maximum 15 degrees below RT) Possibility to match sample and analysis temperature 26 / GE Biacore T100 system presentation / 3/28/2008 6/

7 Racks and vials Both 96 and 384 well microplates can be used Number of samples will sometimes be limited by reagent volume available Both reagent racks from S51 plus additional combined sample & reagent rack (for 7,11,16 mm vials) New plastic 4 ml vial (15 mm ) allowing bigger capture volumes Can also be used in S51 27 / GE Biacore T100 system presentation / 3/28/2008 Support for LMW compound interactions Assay development, analysis and evaluation of LMW interactions Software support MW detection >ca 100 Da (ligand dependent) DMSO solvent correction Azolsulfamide (543 Da) µm binding to Carbanhydras 28 / GE Biacore T100 system presentation / 3/28/2008 7/

8 29 / GE Biacore T100 system presentation / 3/28/2008 Use software wizards for fast assay development, analysis and evaluation Guidance for every interaction parameter Immobilization Assay Development Control Experiments Assays for Binding Analysis (How specific?) Kinetics & Affinity (How fast/strong?) Concentration (How much?) Thermodynamics (Why this fast/strong?) 34 / GE Biacore T100 system presentation / 3/28/2008 8/

9 Immobilization support for various chemistries and Sensor Chip types Pre-defined methods for standard immobilization chemistries Amine Thiol Aldehyde Maleimide Support for customized immobilization methods 35 / GE Biacore T100 system presentation / 3/28/2008 Software wizards simplify kinetic and affinity analysis Wizards guide you step by step Support for direct binding and capture format Sample id and position can be imported from LIMS system 36 / GE Biacore T100 system presentation / 3/28/2008 9/

10 A few simple clicks to complete kinetics evaluation Data fitted to selected interaction model All kinetics parameters displayed within seconds 37 / GE Biacore T100 system presentation / 3/28/2008 One click to display results Choose linear or 4 parameter fitting Calculated conc. and CV% Calibration curve 39 / GE Biacore T100 system presentation / 3/28/ /

11 Transition state thermodynamics Affinity analysis = EQUILIBRIUM thermodynamics Kinetic analysis = TRANSITION STATE thermodynamics Provides additional information about reaction mechanism: Entropy ΔS Enthalpy ΔH Gibb s free energy ΔG Kinetics at varying temperatures 40 / GE Biacore T100 system presentation / 3/28/2008 Automatic Eyring and van t Hoff plots for thermodynamic parameters Display of: Entropy (ΔS) Enthalpy (ΔH) Gibbs free energy (ΔG) For equilibrium and transition state thermodynamics 41 / GE Biacore T100 system presentation / 3/28/ /

12 Biacore T100 system T100 The new generation Biacore system Software Ease of use Flexibility Hardware Visibility Interactivity Robustness Consumables Sensor chips Buffers Kits GxP Support GxP Software GxP Documentation Training material Getting started Basic course LMW course Kinetics & Affinity analysis Service T100 Service contract IQ/OQ/IPQ User Documentation Instrument Handbook Software Handbook 49 / GE Biacore T100 system presentation / 3/28/ /