1. HTRF ® ACTIVE GLP-1 CELL-BASED ASSAY Sales training JULIE VALLAGHE CATHY DREXLER JANUARY 2013 V0 APRIL 2013 V0 Metabolic biomarkers / Diabetes investigation

2. 2 Physiological functions of GLP-1: HTRF Active GLP-1 assay (V0) GLP-1 = Glucagon-like Peptide-1 Intestinal hormone secreted by enteroendocrine L-cells in response to nutrient ingestion. Inactive full-length GLP-1 peptides (1-36) amide and (1-37) are produced by posttranslational processing of proglucagon and are converted into bioactive forms (7-36) amide and (7-37) by prohormone convertase 1/3. GLP-1 potentiates insulin secretion, reduces food intake and inhibits glucagon secretion. Rapidly degraded in the circulation by the dipeptidyl peptidase IV enzyme (DPP-IV). Targeted therapeutic areas: Type-2 diabetes Obesity  Screening of DPP-IV inhibitors or molecules which increase the endogenous release of GLP-1

3. 3 Active GLP-1 assays HTRF Active GLP-1 assay (V0) Competition status Assay format Active GLP-1 form detected Applications LDL / Assay range Price Perkin Elmer (AlphaLISA) HomogeneousOnly (7-36) amideCell-based 27 pg/mL / 27 to 30000 pg/mL 1 296 € (500 tests) Meso Scale Discovery Homogeneous 100% (7-36) amide 31% (7-37) Cell-based Serum /plasma 0.12 pg/mL / 0.12 to 1000 pg/mL 300 € (96 tests) Millipore (ELISA) HeterogeneousAll active forms Cell-based Serum /plasma 6.6 pg/mL / 6.6 to 330 pg/mL 450 € (96 tests) CisbioHomogeneousAll active formsCell-based 25 pg/mL / 43.8 to 1400 pg/mL 1 150 € (500 tests) With this new kit we now provide the 1 st homogeneous cell-based assay to quantify all active forms of GLP-1 Confidential

4. 4 HTRF ® active GLP-1 assay principle HTRF Active GLP-1 assay (V0) TR-FRET

5. 5 HTRF ® active GLP-1 assay protocol HTRF Active GLP-1 assay (V0) 10 µL of HTRF ® conjugate pre-mix + 10 µL of cell supernatant (or GLP-1 standard) ON at 20-22°C Tb 3+ Cryptate- conjugate d2-conjugate 384-well SV white plate Add conjugates before sample Use white microplates Set up reader for Terbium Cryptate Direct assay on cells is not possible (i.e. transfer is mandatory)

6. 6 Kit packaging, part# and pricing HTRF Active GLP-1 assay (V0) Product designationPart number Active GLP1 kit 500 tests 62GLPPEG Active GLP1 kit 5,000 tests 62GLPPEK  Included in each kit: - 1 vial of anti-GLP-1-Tb cryptate - 1 vial of anti-GLP-1-d2 - 1 vial of GLP-1 (7-36) amide standard - 1 vial of Diluent #4 - 1 vial of Reconstitution buffer #5 - 1 package insert  20µL basis Store the kits at -20°C or below upon reception

7. 7 HTRF ® active GLP-1 assay characteristics HTRF Active GLP-1 assay (V0) GLP-1 standard curve Standard curve fitting with spline point-to-point (e.g. on GraphPad Prism). The assay may not be used for the assessment of GLP-1 in serum or plasma samples

8. 8 - HTRF Active GLP-1 assay detects equally well both (7-36) amide and (7-37) active GLP-1 peptides. - Cross-reactivity to intact GLP-1 forms [(1-36) amide and (1-37)] is very low (< 0.4%). - Degraded fragments of GLP-1 as well as GLP- 2 and Glucagon remain undetectable. HTRF Active GLP-1 assay (V0) PeptideCross-reactivity GLP-1 (7-36) amide100% GLP-1 (7-37)98.4% GLP-1 (1-36) amide0.39% GLP-1 (1-37)0.37% GLP-1 (9-36) amide< 0.01% GLP-1 (7-17)< 0.01% GLP-2< 0.01% Glucagon< 0.01%  The assay is highly specific for active forms of GLP-1. Specificity for active GLP-1

9. 9 Detection kinetics HTRF Active GLP-1 assay (V0) Equilibrium reached ON / signal stable over 24h

10. 10 Reader compatibility HTRF Active GLP-1 assay (V0)  Not compatible with Spectramax M5e (Flexstation)

11. 11 Cell-based assay validation HTRF Active GLP-1 assay (V0) GLP-1 secretion assay on glucose-stimulated NCI-H716 cells Human NCI-H716 enteroendocrine cells were seeded in a 96-well culture plate coated with Matrigel. After 2 days at 37°C and under 5% CO 2 atmosphere, cells were washed and stimulated with increasing concentrations of glucose in DPPIV inhibitor supplemented KRB. After 1h stimulation, supernatants were collected and quantified using HTRF GLP-1 assay. As expected, active GLP-1 secretion was induced in a glucose dose-dependent manner.

12. 12 Important guidelines for cell-based assay (P. Ins. excerpt) HTRF Active GLP-1 assay (V0)

13. 13 Diluent #4 must be used with the active GLP-1 assay HTRF Active GLP-1 assay (V0) Same results in diluent #4 and in KRB supplemented with Tween and Triton GLP-1 sticking in KRB without additives !!! GLP-1 standard curve in diluent #4 (of the kit) vs. KRB vs. KRB + Tween + Triton

14. 14 Side-by-side comparison of HTRF and ELISA on NCI-H176 cell supernatants HTRF Active GLP-1 assay (V0) Supernatants containing different levels of GLP-1 were collected from NCI-H716 cells. They were quantified using either the HTRF assay or a fluorescent ELISA kit (Millipore, #EGLP-35K). Assay workflows illustrate clearly the advantages brought by the mix- and-read HTRF process when compared to ELISA. HTRF protocol includes only two addition steps, with one single incubation, whereas ELISA needs 5 dispensing steps, 3 plate washes, and 4 incubations.

15. 15 Side-by-side comparison of HTRF and ELISA on NCI-H176 cell supernatants HTRF Active GLP-1 assay (V0) Correlation between the two methods GLP-1 concentrations were determined for 9 undiluted cell supernatants with each method. Samples 7 to 9, containing the highest levels of GLP-1, could not be quantified with the ELISA kit, demonstrating that the HTRF assay range is more suitable for cell-based assays. HTRF and ELISA quantifications done on cell supernatants 1 to 6 are strongly correlated. [GLP-1] (pg/mL) (+/- 2SD) Cell supernatant HTRFELISA 1 38 (+/- 5)42 (+/- 4) 2 39 (+/- 19)46 (+/- 8) 3 78 (+/- 14)72 (+/- 3) 4 99 (+/- 9)96 (+/- 1) 5 188 (+/- 6)216 (+/- 10) 6 301 (+/- 4)282 (+/- 12) 7 384 (+/- 12) Out of range 8 729 (+/- 76) 9 1302 (+/- 65)

16. 16 Dilution linearity assessment HTRF Active GLP-1 assay (V0) Dilutions of the NCI-H716 cell supernatants 8 and 9 were performed using the HTRF assay diluent. GLP-1 concentration was then determined in undiluted supernatants and in the four corresponding diluted samples (1:2, 1:4, 1:8 and 1:16). Results correspond to the means (+/- SD) of 3 independent dilution tests.  The strong correlations obtained between measured and expected GLP-1 concentrations demonstrate the linearity of dilution within the assay range.

17. 17 Marketing collateral at launch HTRF Active GLP-1 assay (V0) New flyer on assays for diabetes investigation SLAS Poster Kit insert Product web page / Diabetes heroe

18. 18 Conclusions / value proposition HTRF Active GLP-1 assay (V0)  The 1st homogeneous assay to quantify both (7-36) amide and (7-37) active forms of GLP-1 in cell supernatant.  High specificity for both GLP-1 bioactive forms  Dilution linearity of cell supernatants  Mix & read and straightforward protocol: an alternative to conventional technologies such as ELISA  Validation on the human NCI-H716 enteroendocrine cell line, a model commonly used for studying the regulation of GLP-1 secretion  Strongly correlated with ELISA while offering handling easiness, labor time saving, as well as an assay range more suitable for cell-based assays.  A useful complement to other HTRF assays for diabetes investigation, such as insulin.

19. Thank you