Scott Zhou, North China FAS MB: 15810470035 , Email: scott_zhou@ap.pall Mar.20 th , 2013

1. Octet Training Part II: Kinetics on the Octet Scott Zhou, North China FAS MB:15810470035, Email:scott_zhou@ap.pall.comMar.20th, 2013

2. Agenda • Basic Kinetics • BLI Kinetics Workflow • BLI Kinetics Applications

3. Basic Kinetics

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

5. Introduction to Basic Kinetics • Definitions of ka, kd, KD, and kobs • Calculation of ka, kd, KD, and kobs • Concentration Dependent vs. Concentration Independent Parameters

6. ka Ab + Ag Complex kd kobs – kd [Conc Ag (M)] ka= kd ka KD = Binding Kinetics: Overview Our analysis follows a 1:1 binding model: ka = rate of association or “on-rate”; unit = M-1sec-1 kd= rate of disassociation or “off-rate”; unit = sec-1 Y=Y0+A(1-e-kobs*t) Y=Y0+Ae-kd*t Non-linear curve fit of data  ,unit = M • Note that ka (or kon) and KD (affinity value) are concentration dependent (M or molar in the units). Kd (or koff) is a concentration independent value. Kd can be used to screen crude samples relative to each other.

7. Real-Time Kinetics Provides Better Binding InformationOver ELISA Techniques • ELISA methods only provide approximations of KD. • Different combinations of Kon and Koff can give the same affinity measurement. • These two compounds appear to have the same KD, however Example B has a 100x tighter dissociation than Example A. For this pharmaceutical application, a tighter dissociation would be preferred. • Washing steps in ELISA methods will remove weak antibody interactions, and thus are not feasible to use for characterization.

8. BLI Kinetics Workflow

9. Getting Started Computer & Instrument start-up Sensor handling Loading of the instrument

10. Getting Started Turning On the system: Turn on the computer. Next, make sure the sensor and sample positions are clear inside the Octet and turn on the power supply. Finally, launch the Octet Data Acquisition software. Octet will initialize and home the optics box. Warming up the Octet The Octet should be turned an hour prior to an experiment being run to allow for the lamp to warm up. Setting up experiment Use 200 µL sample in each well. Pre-wet the sensors in buffer/media (time may vary depending on sensor – consult product insert).

11. Pre-wetting The Sensors Correct Incorrect • Be sure to get the corner of the 96 well plate under the lip of the sensor tray. If the 96 well plate is above the lip, the sensor tray will be out of alignment and the sensors will not be picked up by the optics box properly. • Keep the sensors in the pre-wet plate at all times when running an assay.

12. Inserting the Sensor Tray into the Sensor Tray Holder and the Sample Plate into the Sample Plate Holder 2 3 1 4 1-The sensor tray is “keyed” to fit into the holder in only one way (it looks vaguely like an arrow and it points into the instrument). 2-The Sample Plate holder is marked “A1” (Red Circle) which corresponds with well “A-1” of the 96 well plate. 3-Place the sample plate into the sample plate holder 4-Proper placement of the sensor tray and sample plate in the Octet.

13. Biosensor selectionAccording to application & sample type etc. • 新开发了四种传感器 • Anti-GST Biosensor: 用于含GST标签的蛋白 • NTA Biosensor：用于含His标签的蛋白 • Anti-human Fab-CH1：用于人Fab, F(ab’)2及Ab1~4 • Anti-Flag biosensor：用于含Flag标签的蛋白

14. NH2 H2N Biotin H2N Amine Reactive biosensor Amine Reactive biosensor Amine Reactive biosensor Immobilization Activation EDC/NHS NHS NHS NHS COOH COOH COOH NH2 COOH H2N NH2 NH2 NH2 NH2 Ligand immobilizationAccording to application & sample type etc. （类）共价键偶联方式：结合稳定，无偶联特异性；要求偶联分子纯度高，浓度要求ug/ml级别；偶联的缓冲液不能含有氨基组分（如Tris），不能含有载体蛋白如BSA等（如有则需先替换），特别适合高亲和力检测。 SA, SSA传感器的偶联：中性环境 Streptavidin biosensor NH2 Minimal biotinylation (in solution) Biotin–linker-NHS Immobilization Streptavidin biosensor Biotin AR2G传感器的偶联：酸性环境，pH条件一般需优化

15. Ligand immobilizationAccording to application & sample type etc. AHC, AMC，Anti-GST, Ni-NTA等传感器的偶联 （类）亲和偶联方式：偶联特异性高，偶联结合力较共价键弱；对靶标样品纯度要求不高，粗样可直接用来偶联（如含靶标分子的培养液，裂解液，腹水等）；高亲和力相互作用推荐选择（类）共价键偶联方式。 Capture protein Analyze kinetics

16. AssociationSample & concentration setting-important • Pre-test 1: • Relative high concentration(10*KD/100*KD). • Positive control. • Blank control. • Yes/No binding. Yes samples go next round detection. • Pre-test 2: • Yes samples go this round or KD test directly according to the researcher. • Find the proper concentration range(using a large dilution factor such as 10 or 5 to determine the highest and lowest concentration) if pre-test 2 performed. • KD test: • No less than 4 concentrations should be included in sample dilution series with a dilution factor 2 or 3. • Positive/negative control. • Blank control. • Data quality determined by R/X square in addition to compare with other source data.

17. Kinetics workflow Baseline Baseline Loading Octet Biosensors Binding (nm) Buffer Ligand-Biotin Protein of Interest Association Dissociation 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

18. Biosensor regenerationDepends on ligand & interaction characteristics etc AHC, AMC,Anti-GST, Anti-His的再生 • 再生步骤：在仪器中在线完成，只需在微孔板中额外加一列再生缓冲液buffer。 • 传感器的再生条件：强酸，强碱，高盐缓冲液。 SA, SSA,AR的再生 • SA,SSA,AR等传感器偶联靶标时，主要通过共价连接的方式，结合力强，再生条件不会将靶标洗脱下来，无需重新偶联（Loading）。 • AHC等传感器主要通过特异的亲和将靶标分子偶联至传感器，结合力较共价键弱，再生条件下，靶标将会被洗脱下来，需重新偶联(Loading)。

19. SA, SSA, AR传感器的再生 Association/ dissociation of target protein Original streptavidin sensor surface Binding of biotinylated receptor Target protein removed • 再生效果跟偶联蛋白的稳定性有很大关系。 • 再生条件可根据传感器使用说明推荐的条件进行，也可实验室根据自身样品自行优化再生条件。 Capture protein Analyze kinetics Regenerate

20. AHC, AMC，Anti-His, Anti-GST传感器的再生 Association and dissociation of target protein Original sensor surface Loading with IgG or Fc containing ligand Sensor back to original surface • 再生之后需重新Loading，之后再进行动力学检测。 • 与SA和AR不同，这类传感器再生之后，可偶联不同的靶标。 • 对偶联蛋白本身的稳定性要求不高。 Capture protein Analyze kinetics Regenerate

21. NTA传感器的再生 Recharge with NiCl2 Association/ dissociation or quantitation of analyte protein Original Nickel NTA sensor surface Functional surface target captured • 与镍柱再生类似。 • 再生之后，较于AHC, AMC, Anti-GST等不同的是：需要对传感器Recharge (NiCl2) Target and NiCl2 removed Capture HIS-tagged protein Analyze kinetics Regenerate

22. BLI Kinetics Applications

23. 小分子化合物筛选、亲和力测定

24. Initial Screening of a Focused Library336 compounds screened and processed in one 384 well plate Small molecule screening-Novartis Blue = target sensor Red = Control sensor

25. Hit confirmation kon: 5E4 M-1s-1 koff: 7E-2 s-1 KD: 1.4 uM Positive control Non-binder Negative control Buffer Non-binder Non-binder Aggregator kon: 2E5 M-1s-1 koff: 1E-1 s-1 KD: 613.5 nM kon: 9E2 M-1s-1 koff: 1E-1 s-1 KD: 140.4 uM Aggregator kon: 2E5 M-1s-1 koff: 1E-1 s-1 KD: 597.8 nM kon: 3E3 M-1s-1 koff: 8E-2 s-1 KD: 24.4 uM kon: 8E5 M-1s-1 koff: 6E-2 s-1 KD: 73.9 nM

26. Steady-state analysis is in agreement with Kinetic analysis Steady-state Analysis of Confirmed Hits Furosemide Steady state KD: 1.3 µM Kinetic KD: 1.4 µM Rack1-C6 Steady state KD: 630 nM Kinetic KD: 613.5 nM Rack 1-D10 Steady state KD: 130 µM Kinetic KD: 140.4 µM Rack7-H3 Steady state KD: 66 nM Kinetic KD: 74 nM Rack1-F5 Steady state KD: 560 nM Kinetic KD: 598 nM Rack3-H5 Steady state KD: 26 µM Kinetic KD: 24 µM

27. Small molecule-Protein interaction Furosemide（呋喃苯氨酸） (MW 330D) Each compound run in a titration series of 5 concentrations in duplicate. Acetazolimide乙酰唑胺 (MW 222D) Sulpiride（硫苯酰胺） (MW 341D) All data from one walk away run

28. Small molecule-Protein interaction Sensor type: SSA Buffer: PBS + 1% DMSO 数据来自Beigene Inc.

29. 蛋白-蛋白、蛋白-DNA、蛋白-糖分子

30. Kinetics of Arabidopsis Proteins measured on Octet Despite gross changes in plant growth rate, Octet data demonstrates the kinetic parameters of the mutated kinase are unchanged from wild type. WT Mutant • Octet QK parameters: • Standard 96W plate • Anti-Murine biosensors • 30C, 1000 RPM in MOPS buffer • Loaded anti-GST antibody to capture GST-BAK protein • 15 minute association w/ BRI1 • 15 minute dissociation

31. DNA-Binding Protein Kinetic Analysis on the Octet QK Dissociation in Buffer Protein Biotin- DNA Binding of DNA-Binding Protein to Immobilized Biotinylated ssDNA Rapid Analysis of Binding of Transcription Factors and Other Promoter Elements to Specific DNA Sequences

32. Saccharide-Protein Data from SWU from Glyconex,Taiwan

33. 克隆筛选、抗体筛选、抗体-抗原亲和力测定、抗体配对克隆筛选、抗体筛选、抗体-抗原亲和力测定、抗体配对

34. Antibody Clone selection Using the Anti-Murine Biosensor to Rank Order Clones High Binding of 7 antibody supernatants in DMEM media supplemented with 15% horse serum Able to quickly bin into high, medium and low producing cell lines The Octet can also be used to monitor the purification process in addition to screening of clones Assay took less than 20 minutes to set up and run on Octet QK Medium Low Blank Media

35. Antibody Screening Sensor type：SA Sample：supernatant without dilution. 数据来自军事医学科学院.

36. Ab Screening & Order rank results 数据来自军事医学科学院.

37. Ab-Ag interaction Kinetic analysis of anti-influenza antibodies using a His-tagged Antigen Detection of anti-influenza virus antibodies in diluted serum to a his-tagged HA antigen immobilized on Anti-Penta His Biosensors on the Octet RED Carney, PJ et al. CLINICAL AND VACCINE IMMUNOLOGY, Sept. 2010, Vol. 17, No. 9

38. Screen for Antibody Specificity and AffinityYes / No Binding Can be Visualized Quickly Buffer B-capture 1 B-capture 2 B-capture 3 B-capture 4 MCP-1 (analyte) Detection 1 Detection 2 Detection 3 Detection 4 A small diagnostic company needed a method to screen commercial Ab sandwich pairs against identified biomarkers to develop a bead-based diagnostic platform. Plate layout was set up to screen 4 capture antibodies against 4 detection antibodies. 32 combinations were tested in 1 hour.

39. Data Analysis Tools Can Quickly Identify Successful Pairs 16 antibody pairs screened, with and without MCP-1. All 5 pairs confirmed specificity and did not bind without analyte. Both selected pairs are specific for MCP-1 and do not bind MCP-2, MCP-3 or MCP-4

40. Cases Octet more preferable • 抗体/蛋白-病毒亲和力测定 • 蛋白-细菌亲和力测定 • 蛋白-纳米颗粒亲和力测定 • 蛋白聚合(蛋白-纳米颗粒) • 蛋白-RNA亲和力测定 • 膜蛋白亲和力测定（蛋白-蛋白） • 定量测定（粗制样品） • 中药研究（蛋白-多糖）

41. Antibody-Virus virus1 virus2 Anti-virus antibody virus3 Neg. virus 数据来自 中科院生物物理所。

42. Protein-bacteria interaction steps: baseline-loading(bio-pro)-baseline-asso(E.Coli)-diss.(PBS) Sensor type: SA Buffer: PB + 0.5%BSA + 0.2% tween-20 数据来自中国农业大学。

43. Protein- Q-dot Immobilize protein on SA sensor surfaceand dilute quantum dot in solution as analyte Data fromWuhan institute of virology,CAS

44. Aggregation :protein-nanoparticle interaction • Object: bio-pro vs nanoparticle • Solution : Loading bio-pro + nanoparticle • Background: proteins form fibers in neutral solution, while in acidic solution the nanoparticle promotes fiber formation, both of which couldn’t be detected with ITC or SPR. • Buffer: pH3.0 HAc; • Outcome: good data.

45. Aggregation :protein-nanoparticle interaction Pro-P1 Pro-P2 数据来自中科院高能所。

46. RNA-ProteinRNA immobilized to biosensors. 10uM KD=6.22E-07,R2=0.988 5uM Associated with protein 2.5uM Bio-RNA loading(10nM) 1.25uM 0.625uM • No NSB signal between 10uM protein and blank sensor without bio-RNA(data not showed) • Actually 5min is enough for association step • Compare with BIACORE, no need to regenerate sensors immobilized RNA. • 数据来自于中科院上海生化与细胞所。

47. Protein-RNA Protein immobilized to biosensors. 数据来自军事医学科学院。

48. Membrane protein-protein interaction • Object: pro vs pro • Solution : Loading bio-pro + pro • Background: there is high percentage of detergents in membrane protein samples thus could not be measured on SPR-based system due to bubbles generated by the buffer. • Buffer: assay buffer containing detergents from customer; • Outcome: good data.

49. Membrane protein-protein interaction 0 Ca2+ 100nM Ca2+ 1mM Ca2+ 100uM Ca2+ 数据来自北大医学部。

50. 中药药理研究中的应用 研究背景: • 中药成分复杂，光吸收法、荧光法等无法有效确证其细胞及活体水平研究结果； • 利用传统SPR技术在原理上检测依赖于折光率变化，但很多碳水化合物与buffer折光率一致，不易引起折光率明显变化；进样模式上依赖于微流控系统，成分复杂容易造成系统堵塞； • BLI检测采用非标记技术、在微孔板中检测速度更快、通量更高，为复杂样品的确证提供有力工具。