DISCOVERY BIOLOGYSupport For Drug Discovery

GMP/GLP compliant facility

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GOOD MANUFACTURING PRACTICE GOOD LABORATORY PRACTICE

FIRST CERTIFICATE 2009 2011

CERTIFICATE ISSUED BY The Chief Pharmaceutical Inspectorate The Bureau for Chemical Substances

CERTIFICATE SCOPE• physical and chemical testing• biological testing in quality control of

medicines

• physical and chemical testing, mutagenicity studies

• chemical analyses and clinical chemistry testing

• cytotoxicity tests of chemical substances• bioanalysis and pharmacokinetic studies

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Discovery Biology at Selvita – organizational chart

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Assay Development& Screening Core Laboratory

Cell & Molecular BiologyCore laboratory

DISCOVERY BIOLOGY

Protein ChemistryCore Laboratory

Recombinant protein production

Protein characterization

Structural biology

Assay Development Unit

Screening & Compound Profiling Unit

Compound ManagementUnit

Biobanking / Bulk Tissue Culture Unit

Flow Cytometry Unit

Competencies

Areas of expertise

• Target validation studies

• In vitro Pharmacology

• Disease modelling

• Cancer Signaling

• Cancer Metabolism

• CNS disorders

• Inflammation

In vivo Preclinical PharmacologyUnit

Design and coordination of animal studies

Execution of the in vivo phase at partner’s

animal facility

Analysis

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Integrated Drug Discovery Services

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Target ID validation Hit ID H2L LOPreclinical

candidate selection

Validated HITs Optimized Hits/Leads Optimized Leads Preclinical Candidate

• Integrated Drug Discovery process at Selvita starts from hit

identification and lasts till preclinical candidate selection

• This process can be expanded with target validation accordingly

to the needs of a Partner

• Every step ends with reaching agreed and defined milestones

• Integrated process, according to Partner needs, can also be

custom composed of any blocks

• Selvita provides highly qualified team of scientists and managers

focused on the project plan and most advantageous solutions

H2L – hit to leadLO – lead optimization

• Selection and validation of in vitro cellular disease models

• Validation of target proteins of functional cellular assays

• Established viability/proliferation assays (MTS cell viability assay, BrdU incorporation assay)

• FACS cell cycle analysis

• Mitotic index for evaluation of proliferation levels

• Cell signaling profiling (ELISA, WB analysis)

• Established LDH – cell death assay

• FACS early apoptosis detection assay

• PARP cleavage for detection of apoptosis

• PARP activity colorimetric assays

• Caspase activity – apoptosis assay (labeled substrates)

• Clonogenic survival assay

• Senescence induction assay (in cells β-galactosidase activity assay)

• Combination index for evaluation of synergy

Selvita’s oncology platforms and molecular biology assays

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CANCER METABOLISM STUDIES EPIGENETIC STUDIES

ENZYMATIC STUDIES

In vitro oncology – capabilities

• Functional assays in various monoaminergic

cloned receptors

• Availability of commercially available, stably

transfected cell lines

• In-house generation and validation of cloned

receptors cell lines

• cAMP-Glo assay, Calcium flux

Determination of the affinity of compounds for

receptor and the binding site density (Bmax)

of receptor families and their subtypes in different

tissue or samples

• Radioligand assays using [3H , 14C] ligands;

MicroBeta TriLux scintillation counter

(PerkinElmer)

• Fast development and optimization of

protocols

• Expertise in handling compounds with low

solubility in water and/or high lipophilicity

• Cytotoxicity: Neuronal cell death assays,

Neurotoxicity (MPP+, 6-OHDA, BSO), Excitotoxicity

(NMDA, glutamate)

▪ Gene and protein expression changes

(qPCR, WB, ELISA, Luminex, AlphaLISA, Flow

Cytometry)

▪ Production of inflammatory mediators

(microglia)

▪ Neurogenesis (BrdU)

▪ Tau hyperphosphorylation

Selvita’s CNS – in vitro pharmacodynamic studies

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In vitro CNS – capabilities

GPCRs

CANCER METABOLISM PLATFORM EPIGENETIC PLATFORM

KINASE PLATFORM

Biophysical platform – hit ID/validation

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PROTEIN FLUORESCENCE THERMAL SHIFT (FTS) ASSAY

Life Technologies QuantStudio 6 Real-Time PCR System with Protein

Thermal Shift technology – ΔTm calculation

MST – MICROSCALE THERMOPHORESIS

In solution analysis with Monolith NT.Automated (labelled vs. label-free) – Kd

SPR – SURFACE PLASMON RESONANCE

Immobilized protein label-free analysis with Biacore T200 (protein-protein

interactions and protein-small molecule interactions) – Kd, kinetic

parameters (kon, koff)

ITC – ISOTHERMAL CALORIMETRY

The system directly measures heat released or absorbed during

biochemical binding events, from which it calculates binding affinity (KD),

stoichiometry (n), enthalpy (ΔH), and entropy (ΔS).

RADIOLIGAND BINDING ASSAY3H-, 14C-Labelled

ELISA, AlphaLISA, TR-FRET/LanthaScreen, enzymatic assays etc.

Kit-based and custom assay development

QuantStudio 6 Flex Biacore T200

MicroCal PEAQ-ITC MST Monolith NT.Automated

HTS-mode available

Other assays

Cell metabolism – lipogenesis, glucose uptake etc.

Cell migration

Angiogenesis assays

Ligand binding, receptor activation, receptor binding

Assays specific for monoclonal antibodies – antibody dependent

cell-mediated cytotoxicity etc.

Enzymatic assays, protein-protein and drug-protein interaction

assays

Design of complementary in vitro studies required by the regulatory

authorities, under GMP certification.

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Toxicology in vitro : Genotoxic assays

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Selvita specializes in genotoxic characterization of chemical agents

Bacterial Reverse Mutation Test(AMES MPF™ Penta I, OECD 471)

The test is a standard bacterial assay which measures histidine reversion

(his- to his+) induced by chemicals causing base changes or frame shift

in the genome of this organism.

In Vitro Mammalian Cell Micronucleus Test (MNA, OECD 487)

This in vitro genetic toxicology assay is performed in the eukaryotic cell line

(CHO-K1) and measures formation of chromosomal changes following

DNA damage induced by the tested agents. This test is used for screening

purposes.

In Vitro Mammalian Cell Gene Mutation Tests Using

the Thymidine Kinase Gene (MLA, OECD 490)

This test is used to detect gene mutations induced by chemical substances

in vitro in mammalian cells. The test measures mutations in thymidine

kinase (TK), which enables to detect different spectra of genetic events. It

constitutes an excellent supplement for the Ames test. This test is used for

regulatory purposes.

Toxicology in vitro: other tests

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Phototoxicity – mouse fibroblasts 3T3 NRU PT (OECD 432)

Skin irritation – Reconstructed Human Epidermis

– EpiDerm™ SIT (EPI-200) method (OECD 439)

Skin corrosion – Reconstructed Human Epidermis

– EpiDerm™ COR (EPI-200) method (OECD 431)

Eye irritation – Reconstructed human Cornea-like Epithelium (RhCE)

(OECD 492)

UV/IR Skin Irradiation, Photo Aging

– determination of anti-oxidant effects,

anti-inflammatory effects, photo-aging prevention

• Cells: primary human epidermal keratinocytes, primary human dermal

fibroblasts

• Measurements: ROS generation, DNA damage and repair,

Gene expression levels (eg. MMPs, Collagen), Cytokine detection using

ELISA/Luminex, Changes in protein phosphorylation (eg. MAPK, p38)

Development of custom, cell-based assays

– based on the literature data and Client’s requirements

ASSAY

DEVELOPMENT

& VALIDATION

Custom assay development service: biochemical and cell-based

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Various detection modes

• absorbance

• fluorescence intensity

• fluorescence polarization

• time-resolved fluorescence (TRF) –

LANCE and DELFIA technologies

Wide range of available cellular models

case-by-case selection of suitable cellular model/s to investigate the biological

mechanism of interest (e.g. cancer cell panels of a specific type and/or with common

mutation patterns), in-house production and validation of recombinant cell lines

(if required)

Multi-factor design of assay development experiments

statistical parameters analysis to support a reliable and robust assay for routine use

• IC50/EC50

• mRSD (mean Relative Standard Deviation)

• S/N ratio

• LanthaScreen

• luminescence measurements

• AlphaLISA, AlphaScreen, AlphaPlex technologies

• SPR

• FACS

ADME/DMPK

In vitro ADME

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Typical secondary ADME(T) tests

• Pharmacokinetic and bioavailability studies

• Metabolite profiling

• Cytochrome P450 inhibition (IC50)

• Genotoxicity and cytotoxicity

(performed by Cell Laboratory – MNA, MLA, Ames)

Typical primary ADME tests

• Solubility (HTS and thermodynamic)

• Lipophilicity (logD estimation at selected pH)

• Cytochrome P450 inhibition (screening)

• Permeability – PAMPA, Caco-2 (CacoReady kit)

• Plasma protein binding

(rapid equilibrium dialysis, ultrafiltration)

• Metabolic and plasma stability

Study type Mouse Rat

Pharmacokinetic

Pharmacodynamic

Pharmacodynamic

In vivo preclinical pharmacology

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Design, coordination and analysis of animal studies

Selvita

• Design• Coordination• Model selection

Partner

• Execution of in vivo phase

• Equipped with modern

infrastructure

• Certified animal suppliers

• Own colonies

Selvita

• Analysis

• Conclusions

• Expertise and tools

for the analysis

Our team successfully designs, coordinates and analyzes data

from studies performed in animals

Selvita expertise and capabilities in:

Pharmacokinetics (in vivo) and analyticsSupportive toxicology testing

Pharmacodynamic studies for confirmation of potency and mechanism of action

In vivo preclinical pharmacology

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Pharmacodynamic in vitro studies

Certified:

Signal transduction analysis

Binding studies and

affinity characterization

Biomarkers analysis in vitro and in vivo

Cytotoxicity and proliferation studies

Development and validation

of biochemical methods

Development and validation

of cell-based assays

Genotoxicity studies

Enzymatic activity testing

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SelvitaKrakow

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THANK YOU FOR ATTENTION!

Get in touch with us: sales@selvita.com