Ghrelin immunoassay & Translational Medicine More information on : http://www.bertinpharma.com/

1. 1 - 04/04/2016
Ghrelin Immunoassay
& Translational Medicine
Stéphane Denépoux, PhD
11th Annual Biomarkers Congress,
Manchester UK
25th & 26th of February, 2016

2. 2 - 04/04/2016
Whatever your molecule…
Bertin Pharma proposes a unique BioAnalysis integrated platform throughout
the drug development process thanks to a long track-record in assay methods
completed in compliant environments.
Biomarker assay application
> soluble protein
> membrane protein
> cell surface antigens
Drug analysis expertise
 NCEs
 peptide
> Protein
> Therapeutic drug antibodies
Immunogenicity assessment (ADA)
> Screening test
> Confirmatory assay
> Neutralising assay
Bertin Pharma handles chemical entities, biologics, peptides and proteins
thanks its highly experimented staff

3. 3 - 04/04/2016
Sample preparation expertise
> Appropriate sampling
> Macromolecules Stabilization in Biofluids
> Validated extraction methods (incl. immunoaffinity)
Ligand binding assays & flow cytometry
> Singulex or Multiplex
> Various detection system
LC-MS/MS MRM powered by MS2Plex
> Highly specific
> High sensitivity (fmol/μg of total protein)
> Simultaneous quantification
> state-of-the art validated assays
Bertin Pharma designs in collaboration with you the best scenario to assay your active compounds
Our customized solutions

4. 4 - 04/04/2016
Whatever your Development Stage
Drug discovery & translational medecine
Non-clinical assay
Clinical trials &
central lab operations
Bertin Pharma is your outsourcing partner whatever the phase you are…

5. 5 - 04/04/2016
Our main Features...
A solid track record
> sustainable background in set-up & validation methods
for NCEs, biologics & biomarkers
> recognized biomarker kit trademarks
Multi-disciplinary teams supported by senior management
> pharmacological or toxicological team
> expert in various analytical methods: Biochemical methods, Hyphenated
methods, etc
Up-to-date means, design & quality
> fit-to-purpose development assay
> update & validation of biomarker assays
> two GLP site facilities

6. 6 - 04/04/2016
 Bertin Pharma Capacities in Translational Medicine
 LC-MS/MS Quantification of DMPK & Oncology Biomarkers
 How Biomarkers evolve with Drug Development Phases
 Presentation of The Ghrelin ImmunoAssay Case Study
Bertin Pharma : Biomarkers & Translational Medicine

7. 7 - 04/04/2016
 human CYP, UGT & Drug Transporters
 MDR-1
 BCRP
 BSEP
 MRP1, MRP2, MRP3
 NTCP
 OCT1, OCT2
 Multiplex assays
 Hepato OATP1B1, OATP1B3, MDR-1, BCRP
 Kidney OCT2, OAT1, OAT3, MDR-1
 BBB MDR-1, BCRP
 Oncology Related Biomarkers
 FAK2, FGFR4, FGFR2, MET, CD33, ERBB3, EGFR, ERBB2,
IGFR, FLT3, VEGFR1, ERBB4, CD37, FGFR1, RET,
PDGFRβ, FAK1, C-KIT, PDGFRα, FGRFR3, VEGFR2
 Others human, mouse or monkey proteins assays to be developed upon request
DMPK & Oncology Biomarkers Currently Available
 OAT1, OAT2, OAT3
 OATP1B1, OATP1B3
 CYP 1A1,1A2, 3A4, 3A5, 2B6,
 CYP 2C8, 2C9, 2C19, 2D6, 2E1
 UGT 1A1, 1A3, 1A6, 1A9, 2B7, 2B15

8. 8 - 04/04/2016
LC-MS/MS Quantification : w/ Pr Tetsuya Terasaki

9. 9 - 04/04/2016
ADME applications: in vitro /ex vivo model validation
in vitro Model Validation
in vitro / in vivo Correlations
Induction / Inhibition Monitoring
Clinical Applications & PBPK

10. 10 - 04/04/2016
 Drug Development Phases ask for different Biomarkers
 Screening
 Animal Studies
 First in Human Studies
 Larger Clinical Trials
 Biomarkers need to be adapted to the Drug Development Stages
 The Ghrelin Case
Biomarkers & Translational Medicine Issues

11. 11 - 04/04/2016
 Resolving unexpected ImmunoAssay challenges
 Dealing with Species Specific issues
 GOAT (Ghrelin Actanoyl Acyl Transferase) inhibitor screening
 Anorexia & renutrition compound screening strategies
 Contribution to constitutional anorexia syndrome description
Ghrelin Immunoassay & Translational medicine

12. 12 - 04/04/2016
 28 Amino Acid Hormone discovered at the same time
by Kojima in Japan & Tomasetto in France
 Produced by Endocrine Stomac Cells & by Hypothalamus
 Present as an Acylated and an UnAcylated form
 Ghrelin O-acyltransferase (GOAT) is responsible for
Acylation.
 The Acylated form stimulates GH-Secretion
Receptors
 Actylated Ghrelin :
 Increases apetite & adipose tissue
generation
 Stimulates neuroendocrine (GH secretion),
metabolic (insulin inhibition) & extra-
endocrine pathways.
Ghrelin : the GH-releasing Hormone

13. 13 - 04/04/2016
 Resolving unexpected ImmunoAssay challenges
 Dealing with species specific issues
 GOAT (Ghrelin Actanoyl Acyl Transferase) inhibitor screening
 Anorexia & renutrition compound screening strategies
 Contribution to constitutional anorexia syndrome description
Ghrelin Immunoassay & Translational medicine

14. 14 - 04/04/2016
 Specific Quantitation of Acylated and UnAcylated Ghrelin is essential to :
 Understand its Biological Fonctions
 Understanding of its implication in Nutrition Disorders (Cachexy, Mental Anorexia, Obesity)
> & for Growth Hormone Secretion and Mobility Disorders
 To support Ghrelin analogs Screenings such as GOAT inhibitors
 Analytical Method Setting up for:
 Screening on Cellular Models
 Screening on Animal Models
> (rodents & non rodents)
 Clinical Trials Support
Ghrelin: 2 forms
 Blood basal level
 Variability between individuals
 Depending on physiological conditions:
> for instance feeding behaviour, …
Ghrelin: Assessment of blood level
 Concentration measured blood sample
Setting-up the Ghrelin ImmunoAssay Kit:
Allowing Ghrelin to become a nutrition research biomarker

15. 15 - 04/04/2016
Setting up of the Ghrelin ImmunoAssay
 Peptide are unstable in biological matrix like blood, plasma or tissue
Other By-product residues (enzymatic, chemical or physical degradation)
Preproghrelin (117 AA)
Desacyl ghrelin
(28 AA)
BuChE
APT-1
Blood
Stomach
Acyl ghrelin
(28 AA+ acylation)

16. 16 - 04/04/2016
Ghrelin assay: How
 Physical or chemical treatment
 Without protection, half time of ghrelin is around few minutes
 Storage at very low temperature (-80°C for instance)
 Use Protease inhibitor
 Wide range of products today available
 More or less specific
 Some of them are toxic
Protease inhibitor :
 Sample treatment have to be compatible with the ImmunoAssay:
 No interference with assay (to be checked for each solution)
Preproghrelin (117 AA)
Desacyl ghrelin
(28 AA)
BuChE
APT-1
Blood
Stomach
Acyl ghrelin
(28 AA+ acylation)

17. 17 - 04/04/2016
Ghrelin assay: one step format
 Sandwich immunoassay in one step for the immunological reaction:
Peptide
(AG or
UAG)
Immunological
Reaction Wash
Detection
Ellman reagent
Detection
antibody
Capture
antibody
 One Step Ghrelin EIA kit:
 One step assay (immunological reaction in one step)
 Use of PHMB (p-Hydroxymercuribenzoate) as Protease Inhibitor
 Evaluation of Protease inhibition activity in vitro in plasma
 Validation for rat/mouse, and human plasma samples

18. 18 - 04/04/2016
Ghrelin Easy Sampling ImmunoAssays
Easy Sampling Ghrelin ImmunoAssay (AG and UAG) EIA kits validated:
 Human, Rat, Mouse, Dog, Pig (undervalidation)
 Validation items:
 Calibration curve: accuracy, CV
 Intra-, inter-run precision and accuracy: 5 levels of QC including LLOQ
 Parallelism, Specificity, Dilution linearity
 Stability: short term, freeze and thaw stability, long term stability
Peptide
(AG or
UAG)
First
Immunological
Reaction
Wash
Detection
Ellman reagent
Detection
antibody
Capture
antibody
Wash
Second
Immunological
Reaction
First Step Second Step
Remove
AEBSF

19. 19 - 04/04/2016
 Resolving unexpected ImmunoAssay challenges
 GOAT (Ghrelin Actanoyl Acyl Transferase) inhibitor
screening
 Dealing with Species Specific Issues
 Anorexia & renutrition compound screening strategies
 Contribution to constitutional anorexia syndrome description
Ghrelin Immunoassay & Translational medicine

20. 20 - 04/04/2016
 Main Objectives & Obstacles
 Acylated Ghrelin Quantification in an in vitro Sample
 Miniaturization / Automation associated with high
performances with less than 1 % false positives
 Primary Evaluated Parameters
 Choice of 384 wells trays
 Choice of Automated Systems
 Operating Conditions Set Up
 Milestones
 Spotting a 25% Inhibition
 Threshold : 188 pg/mL
 <1% False Positives
 > 10 000 Screened compounds
Ghrelin Monitoring : in early Screening Phases

21. 21 - 04/04/2016
 Main Objectives & Obstacles
 Quantification of AG and UAG in PreClinical Species (Mice,
Rats, Pigs, Dogs)
 Establishing Robust Quantification Methods on a very large
Dilution scale (LLOQ: 10 pg/mL to 20 pg/mL)
 Limiting UnAcylation during Sampling and Conservation
 Primary Evaluated Parameters
 Operating Procedures / Sample Management
 Adapting Antibody Couples for each Species
 Optimization of Sample PreTreatment Conditions
 Milestones
 LLOQ : pg/mL for mice/rat/dog, 10 pg/mL for minipigs
 Available Assays : 4 mice/rat, 2 dogs, 2 pigs
Ghrelin Monitoring : in animal Studies
Congrès Int d’endocrinol., 2014
Erasmus University, Alizé Pharma & Bertin Pharma
Peptides 32 (2011) 2274–2282
UMR 894 Inserm/Université Paris Descartes

22. 22 - 04/04/2016
 Resolving unexpected ImmunoAssay challenges
 Dealing with species specific issues
 GOAT (Ghrelin Actanoyl Acyl Transferase) inhibitor screening
 Anorexia & Renutrition Screening Strategies
 Contribution to the Constitutional Anorexia
Syndrome Description
Ghrelin Immunoassay & Translational medicine

23. 23 - 04/04/2016
 Main Objectives & Obstacles
 Sensitivity, Sampling Procedures
 Availability of human Samples for Analytical Validations
 Primary Evaluated Parameters
 Operating Procedures / Sample Management
 Optimization of Sample PreTreatment Conditions
 Assay Validation following Guidelines
 Milestones
 4 ImmunAssay Available
 Clinical Trials sponsored by Alizé Pharma:
> Phase II in Prader-Willi Syndrome (childhood obesity)
> Phase Ib in type 2 Diabetes
> PreClinical Trials in Ischemia / Reperfusion injury
Ghrelin Monitoring : in Clinical Trials
Impossible d’afficher l’image.
Congrès Intl de neuroendocrinol., 2014
UMR 894 Inserm/Université Paris Descartes
Bertin Pharma

24. 24 - 04/04/2016
 J. Epelbaum, D. Grousselles, M.-T. Bluet-Pajot (Inserm U. 549)
 Ghrelin physiology & ImmunoAssay Set Up
 M. Kojima (Nat Cardiovascular Center Res. Institute, Osaka)
 Ghrelin co-founder & Patent owner
 C. Tomasetto (CNRS/Inserm U.184/ULP/ICBMG)
 Ghrelin co-founder & mAb owner
 Atomic Energy Research Center :
 AChE as an ImmunoAssay uniquer marker for EIA
 P. Vitaux & P. Brune (Bertin Pharma)
 ImmunoAssay Kit Production Department
Our International Scientific Network

25. 25 - 04/04/2016
Chemical entities
Let’s Discuss your Projects on :
Biologics
mAbs, Antisense & Fusion Proteins
Advanced Therapy Medicinal Products
Cell, Gene & Tissue Therapies
Vaccines Medical Devices
Cosmetics Nutraceutical
s

26. Thank you for your attention!
For additional details, we remain at your entire service
Take a chance to visit our website:
www.bertinpharma.com
11th Annual Biomarkers Congress, Manchester UK
February 25th & 26th, 2016
Stéphane Denépoux, PhD
Stephane.Denepoux@Bertinpharma.com
Tel: +33 1 39 30 60 68