Will García

Metabolomics & Lipidomics services for the discovery

of new biomarkers.

El Vall d'Hebron Institut de Recerca (VHIR) y la empresa OWL (www.owlmetabolomics.com) han firmado un convenio de

colaboración mediante el cual OWL prestará servicios de metabolómica y lipidómica a los grupos de investigación de este prestigioso instituto de investigación, ampliando así su capacidad de análisis y búsqueda de biomarcadores en un amplio abanico de enfermedades. La Metabolómica es el estudio de pequeñas moléculas denominadas metabolitos, presentes en un sistema biológico. En la actualidad una de las principales estrategias en este campo son los perfiles metabólicos. La comparación de perfiles de distintos fenotipos se utiliza para identificar cambios metabólicos que nos ayudan a conocer la fisiología, toxicología y la forma en la que se desarrolla una enfermedad. OWL posee una potente plataforma de detección de metabolitos fundamentalmente lípidicos, lo cual permite al

investigador multiplicar sus posibilidades de descubrir nuevos biomarcadores y observar detenidamente las alteraciones del organismo tras un determinado estímulo. Los investigadores clínicos y preclínicos de Vall d'Hebron podrán a partir de ahora, no sólo conocer más detalladamente el

fenotipo asociado a multitud de enfermedades en pacientes y modelos animales, sino también

analizar, comparar e incluso predecir la respuesta de éstos ante un determinado fármaco. Esta plataforma de última generación de OWL contribuye a reforzar también los estudios de medicina personalizada de los investigadores de VHIR.

NOTA DE PRENSA: Vall d´Hebron Institut de Recerca y la empresa biotecnológica OWL colaborarán en la investigación para la búsqueda de nuevos biomarcadores de enfermedades.

OWL Metabolomics

To develop diagnostic products and offer

metabolomics services for medical,

pharmaceutical, nutritional and veterinary

research.

MISSION

OWL is a biotechnology company founded in 2002

with its origins in Fatty Liver Disease diagnostics,

using metabolomics.

Introduction

€3 Million equipment investment

Metabolite analysis

= + ?

Metabolic alterations are monitored to have an

overall picture of human or animal health.

Mtb1

Mtb2 Mtb3

Mtb4 Mtb5

CREAM

Introduction

Metabolomics is the analysis of the metabolites that are present in a cell,

tissue or biofluid.

Metabolomics workflow

Experimental Design

LC/MS Analysis

Statistical Analysis

Metabolite Identification

Systems Biology Analysis

Introduction

Multivariate Analysis (MVA) to study metabolic differences

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loading plot: which variables are responsible for

similarity/dissimilarities.

Phospholipids Fatty acids Triacylglycerols Amino acids Steroids Bile acids

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score plot : similarities/dissimilarities

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Statistical Analysis Introduction

Result interpretation Introduction

OWL Technology - Equipment Introduction

Mass spectrometry (MS) based metabolomics offers selective, sensitive analyses with the potential to identify metabolites. The analytical platform at OWL includes several LC-interfaced mass spectrometers: Waters Xevo G2 QTof The Xevo G2 QTof mass spectrometer, with UPLC/MSE and QuanTof technology, offers exceptional levels of sensitivity, selectivity, in-spectrum dynamic range, speed of analysis, quantitative accuracy and exact mass performance simultaneously for any experiment, whether UPLC/MS, MSE or MS/MS. The Xevo G2 QTof is designed to produce accurate quantitative and qualitative information on increasingly large numbers of samples when doing metabolite identification, metabolite profiling, etc. Waters Q-ToF Premier The Q-Tof Premier is a quadrupole orthogonal acceleration time-of-flight (oa-TOF) mass spectrometer that delivers high sensitivity, resolution, and exact mass measurements. The design of this instrument allows MS data to be captured with alternating low-and high collision cell energy. This MSE approach can be used to obtain both precursor and product ion information in the same analytical run to yield the highest confidence in structural elucidation and databank search results. In addition to these features, the Q-Tof Premier can perform MSMS experiments allowing the accurately identification of new metabolites. Waters LCT Premier XE The LCT Premier XE benchtop orthogonal acceleration time-of-flight (oa -TOF) mass spectrometer delivers high sensitivity, resolution, and exact mass measurements for LC/MS applications. Its fast data acquisition rates and automated workflow features match the requirements for MS detection under UPLC conditions. Waters Acquity SQD The ACQUITY SQD combines the resolution, sensitivity, and speed of UltraPerformance technology with single quadrupole MS detection. Waters Acquity UPLC UPLC Technology is a highly robust, dependable, and reproducible system that using sub 2-µm particle technology allows working at higher efficiencies with a much wider range of linear velocities, flow rates, and backpressures to provide optimum chromatographic resolution and sensitivity, essential for high throughput metabolite analysis.

OWL´s 2 Business Lines

Pharma Biotech

CROs Res. Centres

BIOMARKER DISCOVERY SERVICES

Disease progression Drug Mechanism of action Drug efficacy & toxicity Personalized Medicine Dermatology research Nutritional studies

R&D

Ser

vice

s

(ext

erna

l R&

D)

NAFLD*/NASH Test BIOPSY

*NAFLD = Non-Alcoholic Fatty Liver Disease (accumulation of lipid metabolites in the liver)

Dia

gnos

tics

(i

nter

nal R

&D

)

Introduction

Diagnostics

Diagnostic Test

The first in vitro serum-based diagnostic test for steatosis and NASH patient follow-up

Diagnostics

BIOPSY

Other tests in development: •Liver fibrosis and •AST (Alcohol steatohepatitis)

BIOMARKER DISCOVERY SERVICES

R&D Services

Many sample types

Fluids

Serum/plasma Dried Blood Spots (DBS)

Urine Stomach content

Lymph Tear

Tissues

Liver Tissue Kidney Tissue Brain Tissue Lung Tissue

White Adipose Tissue Sciatic Nerve

Stratum Corneum Cornea

Cells Hepatocytes

Oocytes / Spermatozoa Mesenchymal Stem Cells

Hepatoma Cells

Exosomes Hepatocytes Fibroblasts

Breast Cancer Cells Lines

Cult. media Hepatocytes Embryo Culture Media

R&D Services

Over 1200 known metabolites can be analysed.

Lipidomics - Platform 1 • fatty acyls: non esterified fatty acids, oxidised fatty acids and acylcarnitines. • sterol lipids: bile acids and steroids • glycerophospholipids (monoacyl and monoether): phosphoethanolamine, phosphoinositol and phosphocholine • free sphingoid bases

Lipidomics - Platform 2 • cholesteryl esters • sphingolipids: ceramides , monohexosylceramides and sphingomyelins • glycerolipids: diacylglycerols and triacylglycerols • glycerophospholipids (diacyl or ether-acyl): phosphoethanolamine, phosphoinositol and phosphocholine

Amino Acids - Platform 3 • amino acids and amino acid derivatives

Polar Metabolites (CCM) – Platform 4 • Pentose Phosphate Pathway, TCA cycle and Glycolysis • carboxylic acids, nucleosides, vitamins, amino acid derivatives, exogenous or microbial origin • miscellaneous metabolites

Analyses available R&D Services

OWLSkin Platform 1 • fatty acyls: non esterified fatty acids.

OWLSkin Platform 2 • sterol lipids: cholesteryl esters, cholesterol, cholesteryl sulfate. • sphingolipids: ceramides & sphingomyelins. • glycerolipids: diacylglycerols & (triacylglycerols). • glycerophospholipids (diacyl or ether-acyl): phosphoethanolamines, phosphoinositol & phosphocholines.

Specific Platforms for Dermatology Research

R&D Services

Additional analyses R&D Services

Metabolic profiling of non-described pathways

Drug compound tracking (xenobiotic metabolites)

Other analysis and Quantifications

Main expertise: Lipidomics

…at both preclinical & clinical stages

R&D Services

Helps explore many

therapeutic areas..

Recogida y extracción de muestras

Planificación del experimento y recomendaciones de envío de muestras

•Apoyo y guía en la recogida óptima de muestra, procedimientos preparación, metodologías analíticas y procesado de datos.

•Estudiar grupos estadísticamente significativos ya que esta clase de estudios implican la medición de cambios bioquímicos de un grupo respecto a otro. Cantidad mínima de muestras por grupo:

•Cultivo de células: 6 •Animales: 8 •Humanos: 10-20 dependiendo de la variabilidad en cuanto al sexo, edad, etnia…

•Contenedores (microtubos de centrifugación, etc) NO autoclavados. •Identificar cada muestra con un código (En caso de muestras humanas, no deben enviarse datos personales que identifiquen al paciente: nombre, Nº de Seguridad social…) •Enviar además un listado aclaratorio de las muestras. •Las muestras deberán ser enviadas a principios de semana

Alzheimer´s predictive lipids R&D Services

Disease progression markers

Toxicity

Drug efficacy

Patient stratification /Personalised medicine

OWLSkin Health Monitoring

Animal model characterisation

Application examples R&D Services

Autoimmune disease

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Putative biomarker 1 Discriminates between control and patients

Putative biomarker 2 Discriminates between different disease stages

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Autoimmune disease

Progression index = EDSS Disease duration (years)

Example 1: Autoimmune Disease Progression Markers

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R&D Services

Example 2: Toxicity biomarkers

Control B

Control A

Group B+ SS-Galactosamine

Group A+ saline solution (SS)

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Acute liver damage

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core

Metabolic score

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Transaminase levels Metabolic markers

correlate better with apoptosis than

transaminase levels

R&D Services

Example 3: Metabolic profiling in drug efficacy studies

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Day 0: Control Disease induced Treated after dis. induction

Day 8: Control Disease induced Treated after dis. induction

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2 selected markers with opposite trends

M1 M2

R&D Services

Example 4: Patient stratification / Personalized medicine

Study: A rare brain disease Aim: To develop a predictive model for liver abnormalities after administration of X drug. Population: 107 plasma samples from patients ANOVAS + CARTS: 7 models to predict the ALTmax/ALTbasal ratio. In this case, glycerophospholipids were the most relevant

R&D Services

Decreased ceramide level

Normal ceramide level

NORMAL SKIN (normal ceramide levels)

DRY SKIN (decreased ceramide levels)

Allergenic and non-allergenic stimuli

Allergenic and non-allergenic stimuli

Bacteria viruses

Bacteria viruses

A number of skin disorders are related to impairments of the ceramide pattern, which can be measured with OWLSkinTM Platform

Example 5: OWLSkinTM Platforms For Skin Health Monitoring

R&D Services

Example 6: Animal Model Characterisation

Fig. UPLC-TOF base peak ion intensity chromatograms for A)

plasma, B) lymph, C) liver tissue, D) white adipose tissue

and E) stomach content.

R&D Services

A detailed report is given containing:

Experimental methods, data analysis and result interpretation.

Scores plots and diagnostic parameters for the MVA models performed.

Heat-maps.

Raw data, p-value and fold-changes between the different sample groups.

How we work R&D Services

Unique lipidomic capabilities

•Increase your chances of finding biomarkers…

•Exploring areas only recently accessible.

R&D Services

Some of our Customers

Customer Application Drug efficacy markers

Biomarker discovery

Various R&D projects

Mechanistic markers

LEO Pharma Dermatology efficacy markers

Various dermatology projects

Various projects

Ageing markers

Cancer Metabolic biomarkers

Bizkaia Science & Technology Park,, bldng. 502 48160 Derio, Bizkaia, Spain

Will García,

wgarcia@owlmetabolomics.com