Octet Training Part II Kinetics on the Octet

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

Agenda l Basic Kinetics l BLI Kinetics Workflow l BLI Kinetics Applications

Basic Kinetics

Biolayer Interferometry（BLI） 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

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

Binding Kinetics: Overview Our analysis follows a 1: 1 binding model: ka Ab + Ag Complex kd Non-linear curve fit of data ka = rate of association or “on-rate”; unit = M-1 sec-1 kd = rate of disassociation or “off-rate”; unit = sec-1 Y=Y 0+A(1 -e-kobs*t) ka = kobs – kd [Conc Ag (M)] Y=Y 0+Ae-kd*t KD = kd ka , 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.

Real-Time Kinetics Provides Better Binding Information Over 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 100 x 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. Association Dissociation KD = Affinty Example A 1. 00 E+04 1. 00 E-02 1. 00 E-06 Example B 1. 00 E+02 1. 00 E-04 1. 00 E-06

BLI Kinetics Workflow

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

Getting Started • Turning On the system: 1. Turn on the computer. 2. Next, make sure the sensor and sample positions are clear inside the Octet and turn on the power supply. 3. 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).

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.

Inserting the Sensor Tray into the Sensor Tray Holder and the Sample Plate into the Sample Plate Holder 1 2 3 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 “A 1” (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.

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

Ligand immobilization According 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 NH 2 (in solution) Streptavidin biosensor NH 2 Minimal biotinylation Immobilization Biotin–linker-NHS Biotin Streptavidin biosensor H 2 N AR 2 G传感器的偶联：酸性环境，p. H条件一般需优化 Amine Reactive biosensor Activation Amine Reactive biosensor Immobilization Amine Reactive biosensor NH 2 EDC/NHS COOH NH 2 COOH NHS NHS H 2 N NH 2

Association Sample & concentration setting-important • Pre-test 1: 1. 2. 3. 4. • Relative high concentration(10*KD/100*KD). Positive control. Blank control. Yes/No binding. Yes samples go next round detection. Pre-test 2: 1. Yes samples go this round or KD test directly according to the researcher. 2. 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: 1. No less than 4 concentrations should be included in sample dilution series with a dilution factor 2 or 3. 2. Positive/negative control. 3. Blank control. 4. Data quality determined by R/X square in addition to compare with other source data.

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

SA, SSA, AR传感器的再生 Original streptavidin sensor surface Binding of biotinylated receptor Capture protein Association/ dissociation of target protein Analyze kinetics Target protein removed Regenerate • 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。

AHC, AMC，Anti-His, Anti-GST传感器的再生 Original sensor surface Association and dissociation of target protein Loading with Ig. G or Fc containing ligand Capture protein Analyze kinetics Sensor back to original surface Regenerate • 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。

NTA传感器的再生 Recharge with Ni. Cl 2 Original Nickel NTA sensor surface Association/ dissociation or quantitation of analyte protein Functional surface target captured Capture HIStagged protein Analyze kinetics Target and Ni. Cl 2 removed Regenerate • 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 (Ni. Cl 2)

BLI Kinetics Applications

Small molecule screening-Novartis Initial Screening of a Focused Library Blue = target sensor 336 compounds screened and processed in one 384 well plate Red = Control sensor

Hit confirmation Positive control Negative control kon: 5 E 4 M-1 s-1 koff: 7 E-2 s-1 KD: 1. 4 u. M Non-binder Buffer Non-binder Aggregator kon: 2 E 5 M-1 s-1 koff: 1 E-1 s-1 KD: 613. 5 n. M kon: 2 E 5 M-1 s-1 koff: 1 E-1 s-1 KD: 597. 8 n. M kon: 3 E 3 M-1 s-1 koff: 8 E-2 s-1 KD: 24. 4 u. M Aggregator kon: 9 E 2 M-1 s-1 koff: 1 E-1 s-1 KD: 140. 4 u. M kon: 8 E 5 M-1 s-1 koff: 6 E-2 s-1 KD: 73. 9 n. M

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 Rack 1 -F 5 Steady state KD: 560 n. M Kinetic KD: 598 n. M Rack 1 -C 6 Steady state KD: 630 n. M Kinetic KD: 613. 5 n. M Rack 3 -H 5 Steady state KD: 26 µM Kinetic KD: 24 µM Rack 1 -D 10 Steady state KD: 130 µM Kinetic KD: 140. 4 µM Rack 7 -H 3 Steady state KD: 66 n. M Kinetic KD: 74 n. M

Small molecule-Protein interaction Each compound run in a titration series of 5 concentrations in duplicate. Furosemide（� 喃苯氨酸） (MW 330 D) Acetazolimide乙�� 胺 (MW 222 D) All data from one walk away run Sulpiride（硫苯� 胺） (MW 341 D)

Small molecule-Protein interaction KD (M) 2. 34 E 04 KD (M) 3. 73 E 05 kon(1/Ms) kdis(1/s) 3. 09 E+02 R^2 7. 22 E-02 kon(1/Ms) kdis(1/s) 6. 86 E+03 0. 913 R^2 2. 56 E-01 0. 974 KD (M) 8. 41 E-06 KD (M) 1. 04 E 04 kon(1/Ms) 1. 76 E+04 kon(1/Ms) 1. 59 E+03 kdis(1/s) 1. 48 E-01 kdis(1/s) 1. 65 E-01 R^2 0. 985 Sensor type: SSA Buffer: PBS + 1% DMSO 数据来自Beigene Inc. KD (M) 3. 62 E 06 kon(1/Ms) 2. 80 E+04 kdis(1/s) 1. 01 E-01 R^2 0. 980 KD (M) 1. 00 E-05 KD (M) 8. 19 E 06 kon(1/Ms) 1. 18 E+04 kon(1/Ms) 7. 84 E+03 kdis(1/s) R^2 1. 18 E-01 kdis(1/s ) 6. 42 E 02 0. 980 R^2 0. 976

Kinetics of Arabidopsis Proteins measured on Octet WT Mutant Despite gross changes in plant growth rate, Octet data demonstrates the kinetic parameters of the mutated kinase are unchanged from wild type. Octet QK parameters: • • • Standard 96 W plate Anti-Murine biosensors 30 C, 1000 RPM in MOPS buffer Loaded anti-GST antibody to capture GST-BAK protein 15 minute association w/ BRI 1 15 minute dissociation

DNA-Binding Protein Kinetic Analysis on the Octet QK Biotin. DNA Protein Dissociation in Buffer kd ka KD 4. 78 E-05 5. 59 E+0 4 8. 55 E-10 Binding of DNA-Binding Protein to Immobilized Biotinylated ss. DNA Rapid Analysis of Binding of Transcription Factors and Other Promoter Elements to Specific DNA Sequences

Saccharide-Protein Data from SWU from Glyconex, Taiwan

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 Medium Low Blank Media 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

Antibody Screening Baseline Loading Baseline association dissociation buffer Bio-Ag buffer Ab 2 B buffer Bio-Ag buffer Ab 2 D buffer Bio-Ag buffer Ab 1 -1 buffer Bio-Ag buffer Ab 1 -2 buffer Bio-Ag buffer Ab 1 -4 buffer Bio-Ag buffer Ab 2 -1 buffer Bio-Ag buffer Ab 2 -2 buffer Bio-Ag buffer Ab 2 -3 buffer Sensor type：SA Sample：supernatant without dilution. 数据来自军事医学科学院.

Ab Screening & Order rank results Sample ID m. Ab 2 B m. Ab 2 D mono Ab 1 -1 mono Ab 1 -2 mono Ab 1 -4 mono Ab 2 -1 mono Ab 2 -2 mono Ab 2 -3 Respon kdis se kdis(1/s) Error 0. 4546 6. 23 E-03 1. 41 E-04 0. 4178 5. 68 E-03 1. 61 E-04 0. 7806 6. 19 E-03 1. 42 E-04 1. 0178 1. 08 E-02 2. 39 E-04 1. 0235 7. 25 E-03 1. 64 E-04 0. 4227 4. 78 E-03 3. 03 E-04 0. 8906 5. 24 E-03 1. 53 E-04 0. 993 6. 23 E-03 1. 36 E-04 数据来自军事医学科学院.

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

Screen for Antibody Specificity and Affinity Yes / No Binding Can be Visualized Quickly detection 1 detection 2 detection 3 detection 4 B-capture 1 + MCP-1 - MCP-1 B-capture 2 + MCP-1 - MCP-1 B-capture 3 + MCP-1 - MCP-1 B-capture 4 + MCP-1 - MCP-1 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.

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. Capture Ab 1 Both selected pairs are specific for MCP-1 and do not bind MCP-2, MCP-3 or MCP-4 Detection Ab 1 Detection Ab 2 Detection Ab 3 Detection Ab 4 + - - - Capture Ab 2 + - - + Capture Ab 3 + - - + Capture Ab 4 - - B capture 1 MCP-1 Detection 1 B capture 1 MCP- 2 Detection 1 B capture 1 MCP- 3 Detection 1 B capture 1 MCP- 4 Detection 1 B capture 3 MCP-1 Detection 1 B capture 3 MCP- 2 Detection 1 B capture 3 MCP- 3 Detection 1 B capture 3 MCP- 4 Detection 1

Antibody-Virus virus 1 virus 2 Anti-virus antibody virus 3 Neg. virus 数据来自 中科院生物物理所。

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。

Protein- Q-dot Immobilize protein on SA sensor surface and dilute quantum dot in solution as analyte Data from Wuhan institute of virology, CAS

Aggregation : protein-nanoparticle interaction l Object: bio-pro vs nanoparticle u Solution : Loading bio-pro + nanoparticle l Background: proteins form fibers in neutral solution, l Buffer: p. H 3. 0 HAc; l Outcome: good data. while in acidic solution the nanoparticle promotes fiber formation, both of which couldn’t be detected with ITC or SPR.

Aggregation : protein-nanoparticle interaction Pro-P 1 Sample ID P 1 P 1 P 1 P 2 P 2 P 2 KD (M) 2. 95 E-09 1. 22 E-08 kon(1/Ms) 8. 07 E+04 1. 39 E+04 kon Error 5. 91 E+02 1. 06 E+02 数据来自中科院高能所。 Pro-P 2 kdis(1/s) kdis Error 2. 38 E-04 8. 08 E-06 1. 71 E-04 8. 11 E-06 kobs(1/s) 4. 06 E-02 2. 04 E-02 1. 03 E-02 5. 28 E-03 2. 76 E-03 1. 50 E-03 1. 41 E-02 7. 14 E-03 3. 66 E-03 1. 91 E-03 1. 04 E-03 6. 06 E-04 Full R^2 0. 993833 0. 996337

RNA-Protein RNA immobilized to biosensors. 10 u. M 5 u. M Bio-RNA loading(10 n M) Associated with protein 2. 5 u. M 1. 25 u. M 0. 625 u. M KD=6. 22 E-07, R 2=0. 988 • • No NSB signal between 10 u. M protein and blank sensor without bio-RNA(data not showed) Actually 5 min 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。

Protein-RNA Protein immobilized to biosensors. Conc. (n. M) 200 100 50 25 12. 5 KD (M) 2. 97 E-09 kon(1/Ms) 5. 21 E+05 kon Error 4. 41 E+03 kdis(1/s) 1. 55 E-03 数据来自军事医学科学院。 kdis Error 9. 22 E-06 kobs(1/s) 1. 06 E-01 5. 37 E-02 2. 76 E-02 1. 46 E-02 8. 06 E-03 Full R^2 0. 989572

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

Membrane protein-protein interaction 0 Ca 2+ Conc. (n. M) KD (M) kon(1/Ms) kon Error kdis(1/s) kdis Error kobs(1/s) Full R^2 100 n. M Ca 2+ Conc. (n. M) KD (M) kon(1/Ms) kon Error kdis(1/s) kdis Error kobs(1/s) Full R^2 2. 5 2. 14 E-08 2. 75 E+06 8. 04 E+04 5. 89 E-02 1. 32 E-03 6. 58 E-02 0. 934619 2. 5 1. 61 E-08 3. 66 E+06 8. 60 E+04 5. 88 E-02 1. 07 E-03 6. 79 E-02 0. 959003 5 2. 14 E-08 2. 75 E+06 8. 04 E+04 5. 89 E-02 1. 32 E-03 7. 26 E-02 0. 934619 5 1. 61 E-08 3. 66 E+06 8. 60 E+04 5. 88 E-02 1. 07 E-03 7. 71 E-02 0. 959003 10 2. 14 E-08 2. 75 E+06 8. 04 E+04 5. 89 E-02 1. 32 E-03 8. 64 E-02 0. 934619 10 1. 61 E-08 3. 66 E+06 8. 60 E+04 5. 88 E-02 1. 07 E-03 9. 54 E-02 0. 959003 20 2. 14 E-08 2. 75 E+06 8. 04 E+04 5. 89 E-02 1. 32 E-03 1. 14 E-01 0. 934619 20 1. 61 E-08 3. 66 E+06 8. 60 E+04 5. 88 E-02 1. 07 E-03 1. 32 E-01 0. 959003 40 2. 14 E-08 2. 75 E+06 8. 04 E+04 5. 89 E-02 1. 32 E-03 1. 69 E-01 0. 934619 40 1. 61 E-08 3. 66 E+06 8. 60 E+04 5. 88 E-02 1. 07 E-03 2. 05 E-01 0. 959003 80 2. 14 E-08 2. 75 E+06 8. 04 E+04 5. 89 E-02 1. 32 E-03 2. 79 E-01 0. 934619 80 1. 61 E-08 3. 66 E+06 8. 60 E+04 5. 88 E-02 1. 07 E-03 3. 52 E-01 0. 959003 1 m. M Ca 2+ 100 u. M Ca 2+ Conc. (n. M) KD (M) 2. 5 5 1. 73 E-08 kon(1/Ms) 2. 84 E+06 kon Error 6. 28 E+04 kdis(1/s) 4. 93 E-02 kdis Error 8. 14 E-04 kobs(1/s) 5. 64 E-02 6. 35 E-02 Full R^2 Conc. (n. M) KD (M) kon(1/Ms) kon Error kdis(1/s) kdis Error kobs(1/s) Full R^2 0. 959743 1. 25 7. 59 E-09 3. 33 E+06 1. 04 E+05 2. 53 E-02 4. 72 E-04 2. 94 E-02 0. 893988 0. 959743 2. 5 7. 59 E-09 3. 33 E+06 1. 04 E+05 2. 53 E-02 4. 72 E-04 3. 36 E-02 0. 893988 2. 53 E-02 4. 72 E-04 4. 19 E-02 0. 893988 10 1. 73 E-08 2. 84 E+06 6. 28 E+04 4. 93 E-02 8. 14 E-04 7. 77 E-02 0. 959743 5 7. 59 E-09 3. 33 E+06 1. 04 E+05 20 1. 73 E-08 2. 84 E+06 6. 28 E+04 4. 93 E-02 8. 14 E-04 1. 06 E-01 0. 959743 10 7. 59 E-09 3. 33 E+06 1. 04 E+05 2. 53 E-02 4. 72 E-04 5. 85 E-02 0. 893988 40 1. 73 E-08 2. 84 E+06 6. 28 E+04 4. 93 E-02 8. 14 E-04 1. 63 E-01 0. 959743 20 7. 59 E-09 3. 33 E+06 1. 04 E+05 2. 53 E-02 4. 72 E-04 9. 18 E-02 0. 893988 80 1. 73 E-08 2. 84 E+06 6. 28 E+04 4. 93 E-02 8. 14 E-04 2. 77 E-01 0. 959743 数据来自北大医学部。

Summary Workflow Keys & Optimization • Select biosensors • Set proper control(pos. & neg. control) • Pre-wet sensors • • Prepare samples Know KD from publications or other experiment and etc. • Assay buffer optimization(BSA & Tween-20) • Change sensor type • Pre-test 1(Yes/No binding, High concentration-10/100 fold KD, Pos. cont. Screening) • Pre-test 2(determine concentration dilution range) • KD test(no less than 4 conc. ) • KD calculation

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