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Protagen AGOtto-Hahn-Str. 1544227 Dortmund+49-231-9742 6300
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1. Introduction to Protagen AG
2. About Autoantibodies
3. Technology Platform
4. UNIarray® Workflow
5. Protagen Dx Programs – Multiple Sclerosis
6. Protagen Dx Program Overview
Agenda
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1000 m² Modern Lab Space in the
Technology Center Dortmund
Otto-Hahn-Str.15
44227 Dortmund
Germany
Established in 1997
Private Stock Corporation
36 Employees
Protagen AG – Facts and Location
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Protagen AG - Business Units
Protein Services Diagnostics
UNIarray® Technology
High throughput protein expression &
production
Development of novel in-vitro diagnostics
based on autoantibody signatures
R&D focus on diagnostics for MS, RA,
and Prostate Cancer
Platform for companion diagnostics &
vaccine development
UNIchip® Protein Microarrays
Antibody specificity analysis
Antibody ranking
Antibody performance prediction
GMP-compliant Protein Analysis Complete protein characterization Glycans & PTM Impurities & HCP Bioassays
Customized method development
Biomarker & target discovery
Customized Bio-IT Solutions IT support for Protein analytics and Protein
Mass Spectrometry Array data analysis Statistical methods for biomarker
discovery Modiro® – The PTM Explorer ProteinScape®
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1. Introduction to Protagen AG
2. About Autoantibodies
3. Technology Platform
4. UNIarray® Workflow
5. Protagen Dx Programs – Multiple Sclerosis
6. Protagen Dx Program Overview
Agenda
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Arbuckle et al. 2003
Autoantibodies are constitutive and dynamic components of the
immune system change specifically with the development of
diseases and treatment are used for diagnostic purposes are attractive for further biomarker discoveries
Non-disease Disease
Autoantibodies as Diagnostics
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Disease
Responder Non-Responder Adverse effect
Colour Code: Disease Disease Disease Responder Non-Responder Adverse effect
Autoantibodies for Patient Stratification
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Autoantibodies are Superior Markers
Polyclonal AB-Pool
• Measuring autoantibodies in serum or plasma
• takes advantage of the specific polyclonal immune response
directed against immunogenic antigens which become presented
as part of a disease process
• has very high sensitivity of detection of a stable analyte (IgG)
• enables retrospective/prospective studies
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Autoantibodies are Superior Markers
Robust
• polyclonal antibodies are stable in serum for years
• antibody titers do not fluctuate during the day
Easy to obtain from Serum
• minimum invasive procedure
• established procedures in clinical use
Validated as Diagnostics
• used in routine in-vitro diagnostic procedures since decades
• surrogate & non-surrogate markers
• in part established for therapy or disease progression monitoring
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UNIclone® Protein Expression Technology
Largest diversity of recombinant human proteins
> 10,000 different human gene products represented in 38.000 E.coli expression clones
MACROarrays comprising the whole library for screening (E.Coli lysate)
High-throughput protein expression & purification workflow
Proprietary Protein Microarray Production Process
Protagen has exclusive worldwide license from
Max-Planck-Society (Hans Lehrach group), Germany
We utilise…
…a unique Resource for Discovery of Autoantibody Marker Panels
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1. Introduction to Protagen AG
2. About Autoantibodies
3. Technology Platform
4. UNIarray® Workflow
5. Protagen Dx Programs – Multiple Sclerosis
6. Protagen Dx Program Overview
Agenda
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Expression Library Management
Generation, characterization & maintenance of expression libraries
Automated liquid handling and rearraying of clones
Largest library on stock: > 10,000 different human recombinant proteins in > 38,000 expression clones
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High throughput expression of 100 – 1000 of proteins in E. coli/week
Optimized HTS-cultivation in microtiter plate formats
HIS-tag affinity purification, typical yield 150 µg/ml, scalable to low mg levels
Expression in human cell lines and yeast, low µg yields
High-Throughput Protein Expression & Purification
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High-capacity 1D SDS-PAGE for purity control
Automated Capillary Electrophoresis for protein concentration determination using Agilent 5100 ALP
72554334
26
17
130kDa
Protein Characterization/QC
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Contact printing, regular arrays for optimal grid alignment during analysis
Highest flexibility with regard to sample conditions
Capacity: 2 Genetix QArray robots
Biochip Production
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One HS 4800 Pro Station
High throughput automated chip processing
Highest reproducibility, temperature controlled
2 Tecan stations HS 4800 Pro
Capacity: 2 x 24 biochips per run
Biochip Processing
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Fluorescence Read-out Technology
ScanArray Express
Microscope slide formats
4 lasers
High throughput scanning (automated processing of 20 slides)
Broad applicability in microarray scanning
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Bioinformatic Analysis
Interdisciplinary team of biochemists, bioinformaticians, biologists, chemists, biotechnologists
Established algorithms for data normalization and analysis
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1. Introduction to Protagen AG
2. About Autoantibodies
3. Technology Platform
4. UNIarray® Workflow
5. Protagen Dx Programs – Prostate Cancer
6. Protagen Dx Program Overview
Agenda
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Functional Assay: Detection of Patient Autoantibodies
Patient sample on MACROarray
Clone ID:xxx_x14,address in microtiter plate
Polyclonal Autoantibodies (in Patient Serum)
Recombinant protein/ E. coli lysate (MACROarray)
Fluorescent detection cascade (Anti-IgG and secondary labeled Ab)
Serum dilution: 1:20
AP AP
APAssay scheme:
22 x 22 cm PVDF membrane
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• Screening of patient sera on MACROarrays (20 patients)
• Selection of all proteins (Hits) which pass a predefined frequency (i.e. 20% of patient samples express autoantibodies against a certain protein)
• Mixing of all proteins (Hits) with already identified autoantigens from other indications in a customized microarray (current size: 2700x proteins, 3200x design ready)
• Analysis of larger cohorts (n= 50 samples/group), appropriate controls, active controls on 2700x microarrays
• Selection and ranking of best protein marker panels according to statistical significance
• Production of an indication-specific microarray (10-100 proteins)
The UNIarray® -Workflow
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Disease and control samples
Screening of 37,000 human expression clones
Affinity purification of putative biomarkers,
printing protein biochips
Bioinformatical analysis:• Biostatistical ranking• Support vector
machine• Neural nets• Treeboost algorithm• Threshold algorithm…
Patient Sample
Control Sample
Indication-specificbiomarker panel
Diagnostic Protein Array
Disease Non-disease
The UNIarray® -Workflow
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1. Introduction to Protagen AG
2. About Autoantibodies
3. Technology Platform
4. UNIarray® Workflow
5. Protagen Dx Programs – Multiple Sclerosis
6. Protagen Dx Program Overview
Agenda
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Design of the Multiple Sclerosis Study
Multiple Sclerosis Study
Healthy controls
Primary chronic progressive MSInitial manifestation
Secondary chronic progressive MS
Dr. med. Sebastian Schimrigk(new affiliation:Director of NeurologyKlinikum Lüdenscheid)
Clinical partner:
Clinical patient classification according to:
Polman et al., 2005. Diagnostic Criteria for Multiple Sclerosis: 2005 Revisions to the “McDonald Criteria”. Ann Neurol 2005;58:840–846
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Diagnostic Marker Candidates
DNA-Sequences BLASTed & clustered
Number of MACROarray-identified proteins 20 individuals/indication
14
62
9
11
147
110
17 27
58
59
9 23
18
MS
312
9
JIA
190
19
Non-disease
215
RA
272
Indication-uniqueproteins
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Content of Clinical Screening Biochip
Content:
Some 330 top-ranked MS biomarker candidates
were purified and printed in proprietary UNIchip® layout
Analysed samples:
13 non-diseased(7 female, 6 male, mean age: 36.3 y +/- 9.2 y)
30 MS patients (initial manifestation & sec. progr. MS)
(20 female, 10 male, mean age: 39.8 y +/- 8.1 y)
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Signals in relative units,White: normal Blue: positive (cut-off: [mean non-diseased samples + 3 SD; biomarker-specific]) Multiple Sclerosis samples Non-diseased samples
Bio
mar
ker
Quantitative Multiple Sclerosis Protein Biochip
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Color Code: White: normal Blue: positive (cut-off: mean + 3 standard
deviations, biomarker- specific)
Bio
mar
ker
Quantitative Multiple Sclerosis Protein Biochip
Multiple Sclerosis samples Non-diseased
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If suspicious
Protagen MS-IVD
MRI of brain and
spinal cord¹
Lumbar Puncture (CSF²)
Diagnosis
Current Testing for MS:
¹ MRI of value to exclude other diseases, EUR 400-500 / MRI
² Ceresbrospinal fluid
Accurate , 85-95 % sensitivity in trained laboratories,
Inconvenient procedure with , complications such as headache, numbness, infections, bleeding and brain herniation
Clinical Symptoms
Goal:
Replace lumbar puncture
Idea:
Predictive, blood based in vitro diagnostic• better specificity of 90%, • higher sensitivity of 80%
• robust
Multiple Sclerosis still difficult to diagnose….
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SVM analysis using 10-fold cross validation of the currently analysed
samples a high diagnostic sensitivity & specificity is achieved Analysis of 96 CIS patient versus 96 healthy persons (blood donors)
71 % Sensitivity
61 % Specificity
Results of MS Biochip Prototype
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Pre-Product Development Phase Definition of TOP Markers Raw Material Development Assay Development and Optimization
Pre-Validation and Validation Phase Appointment of Clinical Partner(s) - Polyclinic TU Munich, Prof. Hemmer, Germany - Accelerated Cure Project, MA, USA, with collection sites at:
Assay Performance Test on MS Dx
Clinical Validation Study Design
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1. Introduction to Protagen AG
2. About Autoantibodies
3. Technology Platform
4. UNIarray® Workflow
5. Protagen Dx Programs – Multiple Sclerosis
6. Protagen Dx Program Overview
Agenda
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UNIarray®
Indication-specific
Concept
MACROarray
Screening
Antigen
Verification
Serum Screening
Biochip Prototype
•Concept •MACROarray
UNIarray® Projects – Current Status
•Alzheimer D.
•Prostate C.
•Parkinson D.
•Juv. Idiop. Arthritis
•Rheumatoid Arthritis
•Multiple Sclerosis
•Serum Screening
Biochip
•Antigen Verification
• Pancreas C.
•SLE• Breast C.•Ovarian C
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Protagen AG
Otto-Hahn-Str. 15
44227 Dortmund
Germany
T + 49 231 9742 6300
F + 49 231 9742 6301
www.protagen.de
Contact