Transcript Folie 1

Protagen AG
Otto-Hahn-Str. 15
44227 Dortmund
+49-231-9742 6300
Agenda
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
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Protagen AG – Facts and Location
Established in 1997
Private Stock Corporation
36 Employees
1000 m² Modern Lab Space in the
Technology Center Dortmund
Otto-Hahn-Str.15
44227 Dortmund
Germany
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Protagen AG - Business Units
Protein Services
GMP-compliant Protein Analysis
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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®
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
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Agenda
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
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Autoantibodies as Diagnostics
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
Arbuckle et al. 2003
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Autoantibodies for Patient Stratification
Disease
Responder
Colour Code: Disease
Responder
Non-Responder
Adverse effect
Disease
Non-Responder
Disease
Adverse effect
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Autoantibodies are Superior Markers
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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
Polyclonal AB-Pool
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Autoantibodies are Superior Markers
Robust
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polyclonal antibodies are stable in serum for years
antibody titers do not fluctuate during the day
Easy to obtain from Serum
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minimum invasive procedure
established procedures in clinical use
Validated as Diagnostics
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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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We utilise…
…a unique Resource for Discovery of Autoantibody Marker Panels
 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
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Agenda
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
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Expression Library Management
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Generation, characterization & maintenance of expression libraries
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Automated liquid handling and rearraying of clones
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Largest library on stock: > 10,000 different human recombinant
proteins in > 38,000 expression clones
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High-Throughput Protein Expression & Purification
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High throughput expression of 100 – 1000 of proteins in E. coli/week
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Optimized HTS-cultivation in microtiter plate formats
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HIS-tag affinity purification, typical yield 150 µg/ml, scalable to low mg
levels
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Expression in human cell lines and yeast, low µg yields
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Protein Characterization/QC
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High-capacity 1D SDS-PAGE for purity control
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Automated Capillary Electrophoresis for protein concentration
determination using Agilent 5100 ALP
kDa
130
72
55
43
34
26
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Biochip Production
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Contact printing, regular arrays for optimal grid alignment during analysis
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Highest flexibility with regard to sample conditions
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Capacity: 2 Genetix QArray robots
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Biochip Processing
 High throughput automated chip processing
 Highest reproducibility, temperature controlled
 2 Tecan stations HS 4800 Pro
 Capacity: 2 x 24 biochips per run
One HS 4800 Pro Station
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Fluorescence Read-out Technology
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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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Agenda
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
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Functional Assay: Detection of Patient Autoantibodies
Patient sample on MACROarray
Clone ID:
xxx_x14,
address in
microtiter plate
22 x 22 cm PVDF membrane
Assay scheme: Fluorescent detection
cascade (Anti-IgG and
secondary labeled Ab)
Serum dilution: 1:20
Recombinant protein/
E. coli lysate (MACROarray)
Polyclonal
Autoantibodies (in
Patient Serum)
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The UNIarray® -Workflow
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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)
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The UNIarray® -Workflow
Disease and
control samples
Patient
Sample
Disease
Control
Sample
Non-disease
Screening of 37,000
human expression clones
Bioinformatical analysis:
• Biostatistical ranking
• Support vector
machine
• Neural nets
• Treeboost algorithm
• Threshold algorithm…
Affinity purification of
putative biomarkers,
printing protein biochips
Indication-specific
biomarker panel
Diagnostic Protein Array
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Agenda
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
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Design of the Multiple Sclerosis Study
Multiple Sclerosis Study
Healthy controls
Initial manifestation
Secondary chronic
progressive MS
Primary chronic
progressive MS
Clinical partner:
Dr. med. Sebastian Schimrigk
(new affiliation:
Director of Neurology
Klinikum Lüdenscheid)
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
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Number of MACROarray-identified proteins
20 individuals/indication
MS
JIA
312
190
59
147
RA
Non-disease
272
215
11
19
110
9
14
58
9
62
27
17
23
9
Indicationunique
proteins
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DNA-Sequences BLASTed & clustered
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Content of Clinical Screening Biochip
Content:
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Some 330 top-ranked MS biomarker candidates
were purified and printed in proprietary UNIchip® layout
Analysed samples:
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13 non-diseased
(7 female, 6 male, mean age: 36.3 y +/- 9.2 y)
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30 MS patients (initial manifestation & sec. progr. MS)
(20 female, 10 male, mean age: 39.8 y +/- 8.1 y)
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Quantitative Multiple Sclerosis Protein Biochip
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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
Biomarker
Non-diseased samples
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Quantitative Multiple Sclerosis Protein Biochip
Non-diseased
Multiple Sclerosis samples
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Color Code:
White: normal
Blue: positive
Biomarker
(cut-off: mean + 3 standard
deviations, biomarkerspecific)
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Multiple Sclerosis still difficult to diagnose….
Accurate , 85-95 % sensitivity in
trained laboratories,
Inconvenient procedure with ,
complications such as
headache, numbness,
infections, bleeding and brain
herniation
Current Testing for MS:
Clinical
Symptoms
MRI of
brain and
spinal
cord¹
Lumbar
Puncture
(CSF²)
Diagnosis
Goal:
Replace lumbar puncture
Protagen
MS-IVD
Idea:
Predictive, blood based in vitro diagnostic
• better specificity of 90%,
• higher sensitivity of 80%
• robust
¹ MRI of value to exclude other diseases, EUR 400-500 / MRI
² Ceresbrospinal fluid
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Results of MS Biochip Prototype
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SVM analysis using 10-fold cross validation of the currently analysed
samples a high diagnostic sensitivity & specificity is achieved
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Analysis of 96 CIS patient versus 96 healthy persons (blood donors)
71 % Sensitivity
61 % Specificity
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Clinical Validation Study Design
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Pre-Product Development Phase
 Definition of TOP Markers
 Raw Material Development
 Assay Development and Optimization
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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
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Agenda
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
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UNIarray® Projects – Current Status
UNIarray®
Indication-specific
Concept
MACROarray
Screening
•Concept
•Multiple
Sclerosis
•Rheumatoid
•Juv.
 •MACROarray
Antigen
Verification
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•Antigen
Verification
Serum Screening
Biochip Prototype
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•Serum
Screening
Biochip
Arthritis
Idiop. Arthritis
•Parkinson
D.
•Prostate
C.
• Pancreas C.
•Alzheimer
D.
•SLE
• Breast C.
•Ovarian C
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Contact
Protagen AG
Otto-Hahn-Str. 15
44227 Dortmund
Germany
T + 49 231 9742 6300
F + 49 231 9742 6301
[email protected]
www.protagen.de
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