Comprehensive in vitro assays to accelerate biologics characterization and candidate selection with quality, consistency, and speed.
In Vitro Characterization Services
In Vitro Characterization Services • Binding and competition assays • ADC-related assays • T cell engager (TCE) assays • Autoimmune and inflammation assays
In vitro Department provides comprehensive in vitro assays for diverse modalities such as antibodies, fusion proteins, cytokines, BsAbs, antibody-drug
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.
Target Protein Overexpression
Reporter Gene Cell Line
Antigen overexpression
SKOV3-luc-GFP tumor model by IVIS
Ab1 Ab2 Human IgG1
150
Alexa Fluor 647-A- 0.00%
Alexa Fluor 647-A+ 100%
Glo
NF K B Prom.
Luciferase
100
50
Target Cell
0
0.01
0.1
1
10
0
Antibody (nM)
0
10 2
10 3
10 4
10 5
Case Studies Our advanced in vitro 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. 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
0.0001 0.01 1 100 10000 0
0.0001 0.01 1 100 10000 0
10 4
[Abs] nM
[Abs] nM
[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
0.0001 0.01 1 100 10000 0
0
[Abs] nM
[Abs] nM
[Abs] nM
10 3
10 4
10 5
0
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
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 37 C 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)
Conc. (nM) Conc. (nM)
ADC Cytotoxicity
20 40 60 80 100 20 40 60 80 100
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
0.001 0.01 0.1 1 10 100 -20 0 0.001 0.01 0.1 1 10 100 -20 0
Released payload
• • DAR value detection
Target cell
Conc. (nM) Conc. (nM)
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 Internaliza ti on
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)
50
0
0.0001 0.01
1
100
10000
Conc. (nM)
Case Study 3: In Vitro Characterization of TCEs
In vitro
T Cell Killing
Cytokine Production
T Cell Activation
T cell killing to tumor cell TAAhi
Real-time T cell killing assay
IFN- release (Tumor Cell TAAhi + PBMC)
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
Ab1 Ab2 Isotype control
TCE-10nM
0 5
-20 0
hours 0
24
48
72
96
1
100
0.0001 0.01
1
100 10000
0.000001 0.0001 0.01
1
100
Abs (nM)
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-13R ɑ1/I L-4R ɑ bloc ker
• 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
1.5
ligand blocker
Ab1 Isotype control Ligand only Cell only
Ab1 Isotype control
ligand blocker Isotype control
1.0
0.5
0.0
0
0.0001 0.01
1
100 10000
0.01 0.1
1
10 100
10 -3 10 -2 10 -1
10 0
10 1
10 -4
Abs (nM)
Abs (nM)
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
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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