Octet Training Part I The Octet System A

Octet Training Part I: The Octet System， A Label-Free, Real-Time Detection System Scott Zhou, North China FAS MB: 15810470035, Email: scott_zhou@ap. pall. com Mar. 20 th, 2013

Agenda l Label-free & BLI l BLI Applications l BLI Publications l BLI Domestic Users

Label-free & Biolayer Interferometry（BLI）

Labeled：ABS, FI, FRET, Lumi, FP… Label-free?

What is “Label-Free” ? • Label-free is a technology platform that allows monitoring and measuring direct molecular interactions without artificial labels. Why “Label-free”? • Bypass difficulties in label selection and design and avoid artifacts due to the presence of chemical labels • Eliminate use of selected ‘Probes” and the restriction on “probe” binding sites • Direct binding vs. competitive binding • One-step assay

Biolayer Interferometry（BLI）—new generation label-free 什么是光干涉现象

Biolayer Interferometry（BLI） 干涉作用中的相互加强与相互抵消 a b Two waves in phase Constructive interference http: //en. wikipedia. org/wiki/Interference Two waves 180° out of phase Destructive Interference

Biolayer Interferometry（BLI）技术原理 如何利用光干涉现象 White light Relative Intensity Constructive Interference 100% Partially constructive Interference Internal Reference (reflection surface 1 0 Interface with Liquid (reflection surface 2) Destructive Wavelength波� Interference

Biolayer Interferometry（BLI）技术原理 改变膜厚度对干涉光的影响 Relative Intensity 100% 0 Wavelength

Biolayer Interferometry（BLI）技术原理 两个反射表面间距的改变对干涉光强度的影响 Internal Reference (reflection surface 1) Distance between the two reflecting surfaces = Relative Intensity 100% 0 Wavelength (nm) ℓ Interface with Liquid (reflection surface 2) Intensity λ = ƒ(λ, ℓ)

Biolayer Interferometry（BLI）技术原理 可实时检测到两个反射表面间距的改变 Relative Intensity 100% 0 Wavelength (nm) surfaces = ℓ Intensity λ = ƒ(λ, ℓ) nm shift Distance between the two reflecting Time

Biolayer Interferometry（BLI）技术原理 特殊的 BLI • Two-dimensional binding surface 结合在二维的表面 • Biocompatible Matrix - minimizes non-specific binding 具有生物亲和性的表面基团阵列 极小的非特异性结合 • Uniform 一致性高 • Non-denaturing 不会使结合上的蛋白或抗体变性 Biosensor |---- 600 mm ----|

Fortebio Octet 检测平台 Octet QK平台内部

Biosensor selection According to application & sample type etc. 新开发了四种传感器 1. Anti-GST Biosensor: 用于含GST标签的蛋白 2. NTA Biosensor：用于含His标签的蛋白 3. Anti-human Fab-CH 1：用于人Fab, F(ab’)2及Ab 1~4 4. Anti-Flag biosensor：用于含Flag标签的蛋白

BLI Applications & Advantages

BLI Solution for Drug Development Wide Fit Within Discovery and Development Process Research Assay development Expression monitoring Clone selection and ranking Epitope mapping Screening Lead Characterization Preclinical and Clinical Manufacturing and QC Small molecule or ligand screening Affinity measurement Kinetic profiling of immunotherapeutics Production monitoring Kinetic screening of hybridomas On and off-rates Rapid concentration measurements Process optimization Immunogenicity Protein and small molecule therapeutics Secondary hit confirmation Binding affinity ranking 21 CFR Compliant Fast and accurate concentration measurements

Information Provided by Octet Platform Affinity characterization • Binding KD • On and off rates • Rank order Protein Concentration • Picogram to milligram detection range Specificity of binding • Yes / No binding screens • Epitope binning / mapping • Isotyping

BLI Versatility in Interaction Analysis Bacteria� 菌 Size 1000 nm Cells� 胞 200 nm Bacteria� 菌 75 nm Virus病毒 MW Antibody – Antigen抗体-抗原 1, 000 Ig. M免疫球蛋白M Ig. G, Ig. D, Ig. E DNA - Protein 100, 000 Antibody Fragments 抗体片段 1 nm 0. 1 nm Nucleotides (DNA) Small Molecules小分 子 Atoms原子 Antibody Fragment – Antigen抗体片段-抗原 Antigen – Fusion Protein融合入蛋白 Proteins蛋白� Peptides� Receptor – Ligand受体-配体 DNA - DNA Ig. A免疫球蛋白A 7 nm Virus病毒 Antibody – Peptide抗体-� 1, 000 150 1 Multiple Antibody Pairings多重抗体配� Antibody抗体 - Small Molecule Protein 蛋白- Small Molecule

Kinetics workflow Octet Biosensors Buffer Ligand-Biotin Protein of Interest Binding (nm) Baseline Loading Association Time • • 8 or 16 samples can be analyzed in parallel Measure on rates and off rates Data is displayed in real-time Experimental protocols can be customized Dissociation

Quantitation workflow Octet Biosensors Binding (nm) Standards Test Samples • • • The binding rates of test samples are measured and interpolated from the standard curve to determine concentration 96 samples analyzed in 15 - 30 minutes Reuse of standard curve is optional Binding Rate Time (sec) Concentration 120

BLI Publications

Rapid Rise in Publications Citing Octet Platform 200 180 160 140 120 100 80 60 40 20 0 195 141 55 0 2 2006 2 6 2007 7 16 2008 Peer-reviewed Publications 75 48 16 2009 2010 2011/07 All Patents and Publications Since the first Octet system shipment in late 2005, more than 100 articles citing Octet systems have been published in a span of less than 5 years

2012 high IF papers issued till Aug.

Pall Forte. Bio解决方案 u Label-free u Real time u Fluidics-free u Fast，Accurate，Easy. www. fortebio. com