Microarray technology for humoral immune monitoring Johannes Zerweck JPT
2 JPT's Seromarker Discovery Platform Multiplexed biomarker discovery Peptides from HT-SPOT synthesis Multiwell Peptide Microarrays for biomarker verification Peptides from HT-SPOT synthesis Peptide ELISA for validation of detected biomarkers Classical peptide synthesis
3 Production Pipeline Peptide Synthesis, QC and Source Plate Preparation Peptide Cleavage QC by LCMS/MALDI Reformatting of Peptides in Target MTP QC of Target MTP Synthesis Design* Spot-Synthesis *see presentation: „Advanced Library Generation“
4 Ac Membrane Cleavage Quality control Ac Production Pipeline Re-Immobilization of Peptides onto Chips Purified immobilized peptide Chip High-throughput purification Chemoselective immobilization Chip Ac Advantages of JPT Approach All immobilized peptides pass several QC procedures prior printing ensuring highest quality Peptides are immobilized in directed manner resulting in the same and flexible orientation for all peptides All immobilized peptides are purified materials
5 Production Pipeline Microarray Printing and Processing, QC and Assay Workflow Microarray Incubation Experiment Evaluation Microarray Processing Packaging Microarray Printing Microarray QC Cleanroom Facility
6 Assay Principle SeroScreening on Peptide Microarrays = antigenic peptide = fluorophore 1µL of serum (1:200 in Diluent) sec. antibody = antibody Raw data Measurements for each Spot Bioinformatics high-res. scan Evaluation Spot-finding Flagging
7 Seroscreening Experiment Data Processing Automatic processing of tif images with minimal manual intervention desired Raw data file should be ascii table with all relevant information on experiment in header part Evaluation software used at JPT is GenepixPro Result-files so called gpr-files image file (tif)array layout (gal) Evaluation Software automated grid finding feature alignment assignment of sequences Raw data file
12 From Processed-Data to Visualization and Interpretation Heatmap Sequence-Map - residues FASTA-file Sequence-file Pdb-Plots PDB-file Structure-file Sequence-Map - peptides FASTA-file Sequence-file FASTA-file Sequence-file Alignment-Map gpr-file dat-file * * Transformation from.gpr to.dat- file includes calculations of corrected mean values for each triple-set of datapoints.
13 JPT's Seromarker Discovery Platform 1st Module: Discovery High Content Peptide Microarrays Peptides Up to 6.912 peptides in triplicates per slide (total 20.736 peptides) Samples 1 sample per slide Current throughput at JPT 24 samples per day Advantages Low cost per peptide, high peptide throughput Application Broad screen of target antigens on epitope level A limited number of samples (typically 20 to 250) is profiled for primary identification of marker peptides
14 Multiwell Peptide Microarrays Peptides Up to 192 peptides in triplicates per well (21 wells per slide) Samples 21 samples per slide Current throughput at JPT 504 samples per day Advantages Low cost per sample, high sample throughput Compatible with all microtiterplate hardware Application For large number of samples (typically 250 to 1.000) limited set of target peptides For verification of target peptides from primary screening For screening of a biased selection of antigens on peptide levels JPT's Seromarker Discovery Platform 2nd Module: Verification
15 Peptide ELISA Peptides Up to 96 peptides per plate Samples Up to 96 samples per plate Current throughput at JPT 6 plates per day Advantages Economic assay compatible with standard laboratory equipment Chemoselective and directed immobilization of peptides at equal concentrations Robust and reproducible protocols Application For validation of peptide microarray results For development of diagnostic tests As stand alone seroprofiling module for low numbers of target peptides JPT's Seromarker Discovery Platform 3rd Module: Validation
16 Summary PepStar TM Peptide microarrays comprise of chemoselective immobilized purified peptides. Several QC-checkpoints included in production pipeline (LCMS, pipetting controls, process controls, printing controls...), ensuring a controlled quality throughout all production steps. Batch sizes of up to 1000 microarrays per printrun. High Content PepStar TM Peptide microarrays for deep humoral immune response monitoring, epitope mapping & seromarker discovery. Multiwell Array Platform for parallel profiling of hundreds of samples. Peptide ELISA as robust assay platform for confirmation & validation of microarray experiments and development of diagnostic tests.
18 History 1994Jerini Spin-Off from Charité Clinics Berlin 2002 Jerini Peptide Technologies - Business Unit 2004 JPT Peptide Technologies GmbH 2005 JPT Inc. in Boston, MA, USA 2006 New JPT Facilities in Berlin 2009Acquisition by BioNTech AG 2011 50 Employees, 13 PhD‘s Offices & Reps Berlin, Boston, Denver, Brussels 1500 sqm wet lab and 1200 sqm office space Capabilities for organic & biological laboratory work Approved S2 lab for infectious patient samples Cleanroom microarray capabilities Business develoment & marketing History, Quality Assurance & Business Model Quality Assurance 2002 Quality Management System 2004 DIN ISO 9001:2000 Certification Since 2008 GCLP Compliance Audits (NIH, IAVI, BCM, Cellmedica, Genticel...) 2010 DIN ISO 9001:2008 Certification Business Model http://jpt.com http://rnd.jpt.com http://shop.jpt.com
19 Major Application Fields of Peptides Immunology Vaccine development B- and T-cell epitope discovery Diagnostic tools for infectious, autoimmune diseases, allergies & cancer Immune monitoring & immune therapies Proteomics & Enzyme Profiling Enzyme profiling and protein/protein interaction studies Quantification of protein expression levels Biomarker discovery Drug Discovery Biomedical screening Peptide therapeutics SAR, structure activity relationship studies
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