In Vitro Characterization Services

Comprehensive in vitro assays to accelerate biologics characterization and candidate selection with quality, consistency, and speed.

In Vitro Characterization Services • Binding and competition assays • ADC-related assays • T cell engager (TCE) assays • Autoimmune and inflammation assays

In vitro characterization of biologics is crucial for the development of effective biologics candidates. At WuXi Biologics, the CRO Services Department provides comprehensive in vitro assays for diverse modalities such as antibodies, fusion proteins, cytokines, BsAbs, antibody-drug conjugates (ADCs), and more. Leveraging insights from publications, patents, and extensive hands-on experience, our platform efficiently streamlines candidate screening and characterization with exceptional quality, consistency, and speed.

One-Stop, Integrated In Vitro Platform

Binding and Competition Assays • ELISA, FACS, SPR, FRET • Single antigen binding • Dual binding • Receptor-ligand blocking • Epitope binning

ADC-Related Assays

T Cell Engager Assays

Autoimmune and Inflammation

Our integrated suite of in vitro assays allows for a comprehensive evaluation of various targets and therapeutic modalities including antibodies, proteins, cytokines, BsAbs, and ADCs.

• Internalization • Cytotoxicity • Bystander killing • Serum/plasma stability (DAR, payload)

• TCE reporter gene assay • T cell killing

• Immuno reporter gene assay • Cell functional assay • Immune assays • ECD shedding • ADCC, ADCP, CDC

• Cytokine release • T cell activation

Custom Cell Line Development for Bioassays

With over 500 custom cell lines developed, we bring our unparalleled expertise in cell line development to bioassays. Our advanced, tailored solutions use cutting-edge techniques, including random integration, Flp-In system, lentiviral system, and CRISPR knockout.

Target Protein Overexpression

Reporter Gene Cell Line

Target Gene Knockout Cell Line

Antigen overexpression

SKOV3-luc-GFP tumor model by IVIS

Alexa Fluor 647-A- 99.6%

Alexa Fluor 647-A+ 0.441%

Alexa Fluor 647-A- 3.23%

Alexa Fluor 647-A+ 96.8%

Alexa Fluor 647-A- 0.00%

Alexa Fluor 647-A+ 100%

Ab1 Ab2 Human IgG1

150

Glo

NF K B Prom.

Luciferase

100

Target Cell

0.01

0.1

Antibody (nM)

10 2 0

10 3

10 4

10 5

10 2

10 3

10 4

10 5

10 1

10 2

10 3

10 4

10 5

Case Studies Our advanced in vitro platform has proven effective in evaluating challenging molecules and assessing their functional properties. The following case studies highlight key aspects of drug candidate characterization to ensure optimal lead selection.

Case Study 1: Binding and Competition Assays Our binding assays use a range of methodologies, including protein-based assays such as ELISA and SPR, and cell-based assays like cell ELISA, flow cytometry, and FRET, with high-throughput modes available. Additionally, we offer dual-binding analysis for bispecific antibodies and complex interactions, including cell-to-cell, protein-to-protein, and cell-to-protein binding.

High-Throughput Screening: Multiple Cell Lines in ONE Well

1000 2000 3000 4000

15000

30000

Cell-1

Cell-2

Cell-3

Cell line mixture

10000

20000

10 5

5000

10000

Cell-3

Cell-1

Cell-2

0.0001 0.01 1 100 10000 0

10 4

[Abs] nM

200 400 600 800

1000 1500 2000 2500

Cell-4

Cell-5

Cell-6

3000

10 3

2000

Cell-4

Cell-5

Cell-6

1000

10 2

0.0001 0.01 1 100 10000 0 500

0.0001 0.01 1 100 10000 0

[Abs] nM

10 3

10 4

10 5

10 2

BMK1

hIgG1 Isotype Control

hIgG4 Isotype Control

BMK2

Ab1 Ab2

Comp-FarRed-A

Case Study 2: In Vitro Characterization of ADCs

In vitro characterization of ADCs encompasses essential assays, including internalization, cytotoxicity and bystander killing, and plasma stability tests. These assays provide critical insights into the functionality and stability of ADCs.

Internalization

Cytotoxicity and Bystander Killing

Serum Stability

In vitro cytotoxicity assay

Serum/Plasma: human, cyno, mouse and rat

pHrodo

Antigen

mAb screening by hFab-MMAE

20 40 60 80 100 20 40 60 80 100

Ab7.mIgG1K Ab8.mIgG1K Ab9.mIgG2aK Isotype.mIgG1 mFab-MMAE only Ab7.mIgG1K Ab8.mIgG1K Ab9.mIgG2aK Isotype.mIgG1 mFab-MMAE only

Ab7.hIgG1 Ab8.hIgG1 Ab9.hIgG1 Isotype.hIgG1 hFab-MMAE only Ab7.hIgG1 Ab8.hIgG1 Ab9.hIgG1 Isotype.hIgG1 hFab-MMAE only

Late endosome/ lysosome

Target cell

Incubation at 37C for 0/1/4/7/14/21 days

0.001 0.01 0.1 1 10 100 -20 0 0.001 0.01 0.1 1 10 100 -20 0

0.001 0.01 0.1 1 10 100 -20 0 Antibody or ADC 0.001 0.01 0.1 1 10 100 -20 0

anti-FC F(ab)2-pHrodo

Conc. (nM) Conc. (nM)

ADC Cytotoxicity

20 40 60 80 100 20 40 60 80 100

Acid quenching

Ab7.hIgG1 Ab8.hIgG1 Ab9.hIgG1 Ab7.hIgG1 Ab8.hIgG1 Ab9.hIgG1

Antigen

Ab7.hIgG1-MMAE Ab9.hIgG1-MMAE Ab8.hIgG1-MMAE Isotype.hIgG1-MMAE Ab7.hIgG1-MMAE Ab9.hIgG1-MMAE Ab8.hIgG1-MMAE Isotype.hIgG1-MMAE

Acid quench

Isotype.hIgG1 hFab-ZAP only Isotype.hIgG1 hFab-ZAP only

0.001 0.01 0.1 1 10 100 -20 0 0.001 0.01 0.1 1 10 100 -20 0

• Antigen affinity purification • DAR value detection

Released payload

Target cell

Conc. (nM) Conc. (nM)

Lysosome tracking Bound Binding

• Multiple species: human, cyno, mouse and rat • Free-payload release and drug-to-antibody ratio (DAR) can be assessed by LC-MS • Aggregation can be assessed by SEC-UPLC with fluorescence detection • Antigen binding activity (by ELISA/FACS), total ADC and antibody quantification (by ELISA/LC-MS) can be tested to evaluate ADC stability

Internalized Internalization

Bystander killing

Cytotoxicity on Raji.luc (ONE-GLO)

150

100

ADC (Raji-Luc+N87) DXd (Raji-Luc+N87) ADC (Raji-Luc only) DXd (Raji-Luc only)

0.0001 0.01

100

10000

Conc. (nM)

Case Study 3: In Vitro Characterization of TCEs

In vitro characterization of TCEs includes assays for T cell killing, activation, and cytokine release, providing a comprehensive evaluation of TCE efficacy.

T Cell Killing

Cytokine Production

T Cell Activation

IFN- g release (Tumor Cell TAAhi + PBMC)

T cell killing to tumor cell TAAhi

Real-time T cell killing assay

CD69 expression on CD4+ T Cell

100 120

0 20 40 60 80 100 0.000001 0.0001 0.01

10 15 20 25

Isotype -10nM

20 40 60 80

Ab1 Ab2 hlgG4 control

Ab1 Ab2 Isotype control

TCE-10nM

0 5

-20 0

hours 0

100

0.0001 0.01

100 10000

0.000001 0.0001 0.01

100

Abs (nM)

Abs (pM)

Case Study 4: In Vitro Characterization of Autoimmune and Inflammation Molecules

Ligand Competitive Binding

Reporter Gene Assays

Proliferation & Immune Assays

• High-throughput assays • Autoimmune antagonist such as IL-13Rα1/IL-4Rα blocker

• Cellular function screening • Inflammation signaling

• Proliferation assays • Immune assays: T/NK cell activation, cytokine release assays, et al.

Ligand competition assay

IL-31 RGA assay

Inhibition of IL-31-dependent cell proliferation

2 x 10 6 4 x 10 6 6 x 10 6 8 x 10 6 1 x 10 7

2.0

10000 20000 30000 40000 50000 60000 0.0

Ab1, non-eecive ligand blocker Ab2, eecive ligand blocker Isotype control

1.5

Ab1 Isotype control Ligand only Cell only

Ab1 Isotype control

1.0

0.5

0.0

0.0001 0.01

100 10000

0.01 0.1

10 100

10 -3 10 -2 10 -1 10 0

10 1

10 -4

Abs (nM)

About WuXi Biologics

WuXi Biologics is a leading contract research, development, and manufacturing organization (CRDMO) that provides end-to-end capabilities to healthcare organizations worldwide. With operations in China, the United States, Ireland, Germany, and Singapore, we enable our partners to effectively and efficiently bring biologics and vaccines to patients worldwide through our comprehensive and high-quality drug development model.

The world’s leading global single-source platform from concept to commercialization

wuxibiologics.com | PS_BD@wuxibiologics.com

12-3-2024

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