Protein Arrays High Throughput Protein Quantification from Total Cell Lysates

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1 Protein Arrays High Throughput Protein Quantification from Total Cell Lysates Dr. Markus Ehrat Mr. Markus Tobler Zeptosens A Division of Bayer (Schweiz) AG Benkenstr. 254 CH-4108 Witterswil Switzerland

2 Zeptosens at a Glance Zeptosens AG operational, PWG licensed in from Novartis Established production facility for microarrays and readers SensiChip DNA product line launched ZeptoMARK Protein product launched 30 th patent application on bioarrays filed Bayer Technology Services GmbH acquires assets and business of Zeptosens AG

3 Our Expertise: Bioarray Design, Development, Production and Service Infrastructure Clean room and assay development labs DNA and protein microarray production and measurement equipment Biomarker profiling study, data analysis and reporting processes

4 Our Expertise: Bioarray Design, Development, Production and Service Know-how Surface based protein, peptide and DNA assays Established design, manufacturing and quality control processes for microarrays Microarrays image and data analysis Proven Record Developed > 50 protein capture micro array assays Developed > 160 assays for reverse arrays in pharmaceutical analysis Performed > 15 protein profiling studies for pharmaceutical companies Applied > 30 patents on PWG and assay technology

5 Our Expertise: Optical Detection Technologies Infrastructure Optics development labs Final assembly and QC line for PWG readers with CE and ETL certification Process documentation system according to ISO 9001 standards Know-how / network Network to international experts and institutes in optics and microoptics for state of the art development of instrumentation Design and realization of advanced optical concepts CE/ETL certification processes Proven Record Commercial PWG system developed and launched starting from paper concept of technology

6 Zeptosens Product Lines ZeptoREADERTM SensiChip DNA Microarrays ZeptoMARKTM Protein Microarrays ZeptoMARK protein profiling service ZeptoVIEWTM image analysis Assay reagents & protocols

7 Planar Waveguide Principle - for High Sensitivity Fluorescence Microarray Detection

8 Fluorescence Excitation in the Evanescent Field for Highest Detection Sensitivity conventional excitation ZeptoREADER - evanescent detection sample solution support High sensitivity - more information Fast time to result Less sample preparation Direct measurement in blood or serum background problems ~ no background

9 ZeptoREADER Main features of the reader: Fully automated unattended chip readout of up to 360 microarrays Sophisticated control software Excitation lasers: 635nm, 532nm, Highest Sensitivity due to planar waveguide technology (1 zmol) Typical processing time for 30 microarrays: minutes Integrated barcode reader for data traceability One output file per chip containing all image data, chip information and measurement parameters; compatible with image analysis software

10 ZeptoCarrier Integrated microfluidics Automation capability Easy handling Capacity: 6 ZeptoCHIPs / 36 arrays inlet outlet waste chip

11 Protein Microarrays Two Formats Capture Arrays Cell Lysate Arrays Array of target-specific capture molecules (e.g. antibodies) Cell extracts contacted with array Specific detection via labeled secondary Ab (ELISA-type) Two Ab s per target Array of samples Lysate samples spotted Target proteins on chip Specific detection with target-specifc antibodies One Ab per target

12 Capture Arrays

13 Parallel Fluorescence Immunoassay distance from surface pre-incubation mix solution propagating excitation light buffer phase evanescent field chip light intensity h-il2 h-il4 h-il6 waveguide layer glass substrate

14 Quantitative Parallel Assay Needs Selected high quality content: - High affinity Ab pairs - Highly specific Abs - No cross-reactivities Robust parallel assay on the platform Matrix compatibility to achieve good accuracy and precision Sensitive readout - to reach clinical relevant working range of (low) marker conc. - to overcome sample size limitations Assay Validation

15 Performance Cytokine Antibody Array Analyte LOD* LOQ** Dose CV*** Name (all human) Abbr. (pg/ml) (pg/ml) (%) Working range (pg/ml) Granulocyte colony stimulating factor G-CSF Interferon- gamma IFNγ Interleukin-1 beta IL-1β Interleukin-2 IL Interleukin-4 IL Interleukin-6 IL Interleukin-7 IL Interleukin-8 IL Interleukin-10 IL Tumor necrosis factor-alpha TNFα Vascular endothelial growth factor VEGF * Analyte concentration at assay signal of blank plus 2x standard deviation of blank ** Analyte concentration at assay signal of blank plus 8x standard deviation of blank *** Coefficient of variation of dose, determined from standard deviation of calibration curve signal (standard deviation of replicate spot signals) for concentrations > LOQ Shelf life: Comparable assay performance data (LOD/LOQ, Dose CV) after 4 months chip storage at 4 C shown.

16 Breast Cancer Marker Chip

17 Validation of a Parallel Cancer Marker Array 50 human breast cancer cytosols ELISA as a reference upa 100 PAI-1 ZS (ng/ml) ZS (ng/ml) ELISA (ng/ml) ELISA (ng/ml) LOD (ng/ml) LOQ (ng/ml) ZeptoMARK ELISA ZeptoMARK ELISA upa PAI VEGF In collaboration with Oncoscore

18 Reverse Arrays

19 Zeptosens Cell Lysate Arrays From Cells to Protein Profiles # 1...# n Cellular systems Drug treatment Cell lysis Spotting Sample Lysis Spotting ~10 6 cells 50 µl lysis 400 pl spotting ~ 1 mg tissue volume volume

20 Optimized Chip and Array Layout 6 Arrays / Chip Ref Ref Ref Ref Zeptosens Buffer Standards Ref ( ) = reference spots containing constant fluorescence 32 Reference 0.4 Cell lysate (mg/ml) up to 32 samples, in 4 dilutions, in duplicates, or 128 samples, in 1 dilution (not shown) Reference

21 Array Layout 32 samples spotted in duplicates and at 4 concentrations; 4x22 reference spots e.g sample #1 Column with reference spots Line profile (as depicted in image) Fluorescence Reference Reference Distance (pixel) controls

22 Zeptosens Cell Lysate Arrays From Cells to Protein Profiles #1...# n Cellular systems Drug treatment Cell lysis I II Ab2 III Ab3 Ab4 V Readout Ab1 IV V Image Analysis Spotting Assay Ab5 Ab6 Bioinformatics Blocking

23 Multiplexed Monitoring of Signaling Events Treatment Raf Ab Erk2 Ab 6 identical Lysate Arrays P-Erk2 Ab P-Akt Ab Akt Ab Bcl-2 Ab Effect From Cell Signaling Technology

24 Assay and Readout Raf Ab III IV II I II P-Akt Ab Akt Ab Bcl-2 Ab Erk2 Ab V V P-Erk2 Ab VI 8 Akt (Thr202/Tyr204) 8 6 Erk2 P- (Thr202/Tyr204) 8 P-Erk2 (Thr202/Tyr204) C C C TC 4 T C T C T C C T T B 6 C T 1 T 2 C T 1 T C TC T C T C T C 9 T buffer 4 C T T B buffer C T 1 T 2 C T 1 T C TC 4 T C T C T C T C T T B 2 Cell line buffer C T 1 T 2 C T 1 T C TC T C T C T C T C T T B 2 Cell line buffer Erk2 P- Raf (Thr202/Tyr204) 8 Erk2 6 P- 6 Erk2 P- Akt (Thr202/Tyr204) 4 8 Bcl Erk2 P- 2 (Thr202/Tyr204) T1T2 T1T2 2 C T 0 I MEN2A GIST882 Ba/F3 FLT3-ITD MEN2A Ba/F3 bcr-abl KB31 Ba/F3 FLT3-ITD A31 (PDGFR) GIST882 Ba/F3 bcr-abl A431 (EGFR) MEN2A KB31 NWT-21 (IGF-1R) A31 (PDGFR) Ba/F3 FLT3-ITD GIST882 U87-MG A431 (EGFR) Ba/F3 bcr-abl Spotting Buffer MEN2A NWT-21 KB31 (IGF-1R) U87-MG GIST882 Ba/F3 FLT3-ITD A31 (PDGFR) Spotting Buffer Ba/F3 bcr-abl A431 (EGFR) MEN2A KB31 NWT-21 (IGF-1R) Ba/F3 FLT3-ITD A31 (PDGFR) U87-MG GIST882 Ba/F3 bcr-abl A431 (EGFR) Spotting Buffer MEN2A KB31 NWT-21 (IGF-1R) A31 (PDGFR) Ba/F3 FLT3-ITD U87-MG A431 (EGFR) Spotting Buffer Ba/F3 bcr-abl NWT-21 KB31 (IGF-1R) U87-MG A31 (PDGFR) Spotting Buffer A431 (EGFR) NWT-21 (IGF-1R) U87-MG RFI RFI RFI RFI GIST882 Cell line RFI Cell line RFI C T 1 T 2 C T 1 T 2 C TC T C T C T C T C T T B Spotting Buffer Assay Cell line buffer C T 1 T 2 C T 1 T 2 C TC T C T C T C T C TC T B Cell line buffer Microarray & Image Analysis ZeptoREADER TM Protein expression / activation profiles ZeptoVIEW TM

25 ZeptoMARK CeLyA Excellent Correlation with Today s Gold Standard P44/42 MAPK total p-p44/42 MAPK con A B C D con A B C D Fluorescence Index Control A B C D Treatment Fluorescence Index Control A B C D Treatment Good correlation between Western Blot and CeLyA In collaboration with Hoffmann-La Roche

26 CeLyA - Testing Efficacy of Drug Candidates Typical studies comprise: 30 to 60 samples,30 to 80 proteins corresponding 14,400 to 76,800 data points Customer study: Impact of 6 drugs and drug candidates on 20 different signal pathway proteins in 9 different cell lines with 3 different stimulants 5 drugs: 1 concentration 1 drug: 2 different concentrations In collaboration with Novartis Pharma AG

27 β-actin Assay for Array Quality Control (Green) 30 β-actin β-actin level is comparable for all treatments within a cell line All samples were reproducibly spotted Fluorescence signal (10 3 levels) C T 1 T 2 C T 1 T 2 C T C T C T C T C T C T C T B KB31 Cell line buffer GIST882 MEN2A Ba/F3 FLT3-ITD Ba/F3 bcr-abl A31 (PDGFR) A431 (EGFR) NWT-21 (IGF-1R) U87-MG Spotting Buffer

28 Study Results e.g. phospho-erk2 Treatment 8 P-Erk2 (Thr202/Tyr204) 6 RFI 4 2 Effect In collaboration with Novartis Pharma AG From Cell Signaling Technology 0 C T 1 T 2 C T 1 T 2 C T C T C T C T C T C T C T B GIST882 GIST882 MEN2A MEN2A Cell line buffer Ba/F3 Ba/F3 FLT3-ITD FLT3-ITD Ba/F3 Ba/F3 bcr-abl bcr-abl KB31 KB31 A31 A31 (PDGFR) (PDGFR) A431 A431 (EGFR) (EGFR) NWT-21 NWT-21 (IGF-1R) (IGF-1R) U87-MG U87-MG Spotting Spotting Buffer Buffer Highly sensitive multi-parallel protein expression / activation profiling

29 Time-Course Information on Pathway Activation RFI phospho Erk total Erk +staurosporin time after stimulation with αcd3/αcd28 (min) Kinetic profiling (0-60 min) of MAP Kinase activation Jurkat cell lysates upon stimulation with αcd3/αcd28. Levels of phosphorylated and total p44/42 Erk examined using corresponding specific antibodies Array precision allows the quantification of 10-20% changes of phosphorylation

30 Bar Plot Profile for Protein X Cell Line A Cell Line B cell line 1 compound 1 cell line 1 compound 2 cell line 2 compound 3 cell line 2 compound 4 => decreasing concentration => decreasing concentration => decreasing concentration decreasing concentration

31 Tissues Profiling of Human Colorectal Cancers Goal: Identification of markers towards targeted therapy Colorectal cancer samples under well controlled conditions (control, treated) Lysates produced from fresh patient tissues First expression profilings done for 14 (phospho-) proteins Antibodies from (1) Jurkat control (2) Jurkat treated (positive control for up-regulation of Erk2) (3) Buffer (negative control) (4) Tissue lysate, control (5) Tissue lysate, treated In collaboration with Inselspital, Switzerland

32 Tissues Profiling of Human Colorectal Cancers Goal: Identification of markers towards targeted therapy 2.0 fold signal P-Erk2 P-Akt P-p38 P-SAPK/JNK P-IkB-a P-Stat3 alpha-catenin beta-catenin P-Histone H3 P-Rb P-p53 Cyclin D1 Cleaved PARP Cleaved caspase 3 limits of significance First hits of potential markers detected Verification on a larger set of patients ongoing In collaboration with Inselspital, Switzerland

33 ZeptoMARK CeLyA Economic Efficiency Zeptosens CeLyA vs. Western Blot 32 Samples, 4 Dilutions and 100 Target Proteins Cost per Data Point 0.80 EUR EUR 95% lower cost CeLyA WB Total Sample Protein 90 µg 1000 µg 90% less sample CeLyA WB Ab consumption 0.02 ml 0.5 ml 95% less reagent CeLyA WB Time to result 10 times faster 10 days CeLyA 100 days WB