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
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
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 Protein Expression & Purification
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
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Protein Characterization/QC
High-capacity 1D SDS-PAGE for purity control
Automated Capillary Electrophoresis for protein concentration
determination using Agilent 5100 ALP
kDa
130
72
55
43
34
26
17
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Biochip Production
Contact printing, regular arrays for optimal grid alignment during analysis
Highest flexibility with regard to sample conditions
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
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
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:
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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Quantitative Multiple Sclerosis Protein Biochip
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
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
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
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Clinical Validation Study Design
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
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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
•Antigen
Verification
Serum Screening
Biochip Prototype
•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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