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Cell-Based Assays for Biologics

Functional Screening

QC Lot-release

Neutralizing Antibody Assays

US: tel | 1.866.448.4864 e | [email protected] EU: tel | +44.121.260.6142 e | [email protected] www.discoverx.com

Pharmaceutical companies are developing an increasing number of biologics, biosimilars and

biobetters, creating a need for state-of-the-art cell-based assays. DiscoveRx PathHunter bioassays

are highly validated, simple, target-specific cell-based assays that support various stages of bio-

logics development.

Applications For Development of Biologics

R&D Groups – Cellular Binding, Functional Screening and Potency Determination

Manufacturing or Bioanalytical Groups – Bioassays for QC Lot-release and Stability Testing

Clinical Immunology Groups – Cell-based Assays for Detection of Neutralizing Anti-Drug Antibodies

This wide variety of functional readouts gives you complete flexibility to choose the ideal detection and screening platform.

PathHunter Technology

Ligand Ligand

Ligand Ligand

Receptor Assay Pathway Assay

Ligand-dependentDimerization

Binding-basedInternalization

Activation-basedProtein Recruitment

Activation-basedSignaling Event

Types of PathHunter Assays

Applications of PathHunter Assays

Applications of PathHunter Assays. (A) Functional anti-ErbB3 antibodies show a dose-dependent inhibition of signal in PathHunter ErbB2-ErbB3 assay, which detects the recruitment of SH2-domain protein upon activation of the receptor. (B) Anti-ligand antibody against the GLP1 protein demon-strates a dose-dependent inhibition in the PathHunter GLP1R assay, which detects the recruitment of β-arrestin upon activation of the GPCR. (C) A receptor internalization assay with an agonist for OPRD1, a GPCR opioid receptor, which detects internalization of the GPCR to the late endosome. (D) Anti-receptor antibody against the TGF βR2 demonstrates a dose dependent inhibition in the PathHunter TGF βR1 /R2 dimerization assay, which detects the dimerization of the two receptors when bound by their ligand.

10-13 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-50

1000

2000

3000

4000

DADLE [M]

RL

U

Antibody (g/ml)

RL

U

10-10 10-8 10-6 10-4 10-20

200000

400000

600000

800000

1000000

control antibody

anti-Receptor

10-10 10-9 10-8 10-7 10-6 10-5 10-40

20000

40000

60000

80000

Anti-GLP1 Antibody

Negative Control

Antibody [g/ml]

RL

U

10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-40

500

1000

1500

2000

2500

Heregulin

Anti-ErbB3 1

Anti-ErbB3 2

Anti-ErbB3 3

Anti-ErbB3 4

Agonist/Antagonist (g/mL)

Mea

nR

LU

A B C D


PathHunter Applications

Quantify Drug Activity in Lysate, Supernatant and Serum

High tolerance of complex biological samples. PathHunter assays can detect functional biologics in various samples, such as human serum, plasma, hybridoma lysates and bacterial lysates. Above, we validated with GIPR assay with up to 8.5% bacterial lysate, the HRH1 assay with up to 10% normal human serum and were able to detect antibody-based inhibition of the CCL20 ligand in up to 80% serum without any loss of sensitivity.

25000

20000

15000

10000

5000

10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5

8.5%

4.25%

2.13%

0%

10-40

RLU

GIP [M]

Assay in Bacterial Lysate400000

300000

200000

100000

010-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3

Histamine [M]

Assay in Human Serum8000

7000

6000

5000

4000

3000

2000

1000

0

RLU

10-8 10-7 10-6 10-5 10-4 10-3

No FBS

20% FBS

40% FBS

60% FBS

80% FBS

Anti-CCL20 mAb [g/mL] + 10nM CCL20

Anti-CCL20 in Serum

Simple Protocol with Rapid Results

Plate Cells Treat with Agonist/Molecule Read Luminescence

Addition of Detection Reagents

Add compounds

1-24 hours 2-4 hours

One-Step, Plate-Treat-Read Protocol

Superior Bioassay Performance from PathHunter Assays

Target-specific response Inter-lot reproducibilityIntra-lot reproducibility Assay Linearity

10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-40

50000

100000

150000

200000

250000

Vial #1Vial #2Vial #3Vial #4Vial #5

Exendin-4[M]

RLU

10-12

10-11

10-10

10-9

10-8

10-7

10-6

10-5

0

10000

20000

30000GLP1 [M]

Exendin-4 [M]

IGF1 [g/ml]

hGH [g/ml]

Agonist Concentration

RLU

10-12

10-11

10-10

10-9

10-8

10-7

10-6

10-5

10-4

0

100000

200000

300000

400000

Lot 1Lot 2

Exendin-4[M]

Lot 3

RLU

Expected Relative Potency,%0 50 100 150 200

0

50

100

150

200Y = 1.114*X - 9.500R2=0.9986

Mea

sure

d R

elat

ive

to P

ote

ncy,

%

Validation of Analytical Procedures


Select Publications 1. Goldshmit et al., Blockage of Lysophosphatidic Acid Signaling Improves Spinal Cord Injury Outcomes. The American Journal of Pathology, 2012, 181: 978-9922. Foreman et al., ErbB3 Inhibitory Surrobodies Inhibit Tumor Cell Proliferation In Vitro and In Vivo. Mol Cancer Ther, 2012, 11:1411-1420. 3. Santulli-Marotto et al., Surrogate Antibodies That Specifically Bind and Neutralize CCL17 But Not CCL22. Monoclonal Antibodies in Immunodiagnosis and Immunotherapy. 2013, 32: 162-171.

For a complete list of publications of PH applications for biologics visit www.discoverx.com/publications

DiscoveRx 42501 Albrae Street, Fremont, CA 94538 © 2013 DiscoveRx Corporation. All Rights Reserved. DiscoveRx logo and DiscoveRx are registered trademarks of DiscoveRx Corporation. 091213

PathHunter Assay Options

Flexibility of Available Formats

Assay-Ready Kits Stable Clonal Cell Lines Services Custom Assay DevelopmentReady-to-Assay frozen cells Frozen cells (Unlimited culture) Leverage our expertise Access to all technologies

Detection reagents Validated culture reagents Multiple flexible options Expertise from our R&D team

Cell plating media & components High throughput capabilities Experience with 200+ projects

World’s Largest Collection of Cell-Based Assays for Biologics

RTK Assaysc-KIT ErbB4

c-MET FGFR1

c-Ret-GFRα2 IGF1R

DDR1 INSR

DDR2 INSR (Internalization)

EphA4 PDGFRa

EphA5 PDGFRb

EphA7 TrkA

EphB1 TrkA-P75

EphB2 TrkB

EphB3 TrkB-P75

EphB4 TrkC

ErbB1 TrkC-P75

ErbB2-ErbB3

Assays for Multiple GPCR Families Acetylcholine CRHR1&2 MCHR1&2 Prokineticin

Adenosine Dopamine Melanocortin PRLHR

Adrenoreceptor Endothelin Melatonin Prostanoid

Anaphylotoxin Estrogen GRM Protease

Angiotensin Formylpeptide Motilin PTH receptor

Apelin Free Fatty Acid NMUR1&2 Relaxin

Bile Acid GABBR1&2 NPBWR1&2 Serotonin

Bombesin Galanin NPFFR1&2 Somatostatin

Bradykinin Ghrelin NPSR1 Succinate

Calcitonin Glucagon Neuropeptide Y Tachykinin

Calcium-Sensing Glutamate Neurotensin TAAR1

Cannabinoid Glycoprotein H Opioid TRHR

Chemerin GNRHR Orexin UTS2R

Chemokine 5HT Receptors Orphans Vasopressin

Cholecystokinin Histamine Orthologs VIP & PACAP

Class A Orphan HCA Receptors OXGR1

Class B Orphan KISS1R P2Y

Complement Leukotriene QRFPR

Corticotropin LPA & S1P PTAFR

Receptor AssaysTNF receptor

TGFbR1&2

BMP receptors

TLR receptors

IL17 receptors

Frizzled receptor

Cytokine ReceptorsGCSF Receptor

Epo Receptor

GHR

PRLR

>600 GPCR Assays >30 RTK Assays

A subset of the vast number of assays available for biologics development are shown here. For a complete and updated list visit www.discoverx.com/biologicsmenu

Compatible – With complex biological samples

Accurate – Target-specific functional response

Simple – One-step, mix & read protocol

Rapid – Short time to results

Sensitive – Low nM sensitivity

Robust – Precise, linear and highly reproducible

Features and Benefits

cell-based assays for biologics - total solution for bio ... assays for biologics ......

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