High-throughput, high-titer CHO production of miniproteins with high yields and low endotoxin levels, surpassing traditional E. coli production methods.
In-Depth SPR Characterization Throughout Biologics Drug Discovery and Development
Yu Tian, Lei Guo, Jiansheng Wu
WuXiBiologics,No.240HedanRoad, PudongNewDistrict,Shanghai,China (Contact: PS_Marketing@wuxibiologics.com)
Introduction
Case Study 3: High-Throughput Epitope Binning Epitope is a critical determination for antibody functionality. High-throughput epitope characterization methods allow the selection of epitope-diverse molecules early in antibody discovery. Fig 6. 34 × 34 Pairwise Binning by Carterra LSA
Surface plasmon resonance (SPR) is a powerful tool for biologic drug characterization, o ff ering real-time, label-free, and high sensitivity detection of biomolecular interactions. SPR also provides informative data, such as association rate constant (k a ), dissociation rate constant (k d ) and equilibrium dissociation constant (K D ). This technology has been widely applied in the characterization of therapeutic antibodies, antibody-drug conjugates (ADCs) and small-molecule drugs throughout drug discovery and development stages. Fig 1. Key Applications Through Drug Discovery and Development
Binder Non-blocker
• • • • • •
Protein-protein, protein-peptide, protein-small molecule binding a ff inity Bioactivity quality control for protein production
Target A ff inity Characterization Fc Receptor Characterization k o ff Ranking and Screening
Antigen alone control
Full panel of Fc receptors binding for e ff ector function assessment (Fc γ Rs, FcRn and C1q) Fc-engineered antibody characterization k o ff ranking for lead optimization (humanization, a ff inity maturation and developability optimization) High-throughput screening for lead identification (hybridoma and phage screening)
Epitope Binning
•
High-throughput pairwise binning
Simultaneous Binding
•
Simultaneous binding for bispecific antibodies
Biacore 8K/8K+ × 6
Biacore T200 × 3
Carterra LSA × 1
Case Study 4: k o ff Ranking and Screening
Case Study 1: Target A ff inity Characterization
k o ff ranking using crude supernatant Di ff erentiate variants/hits with kinetics data Antibody humanization/PTM removal/a ff inity maturation
High-throughput hybridoma crude sample testing
Successfully developed SPR binding for 200+ targets Multi-pass transmembrane protein binding Fig 2. Target Binding A ff inity Determination
100 μ L per crude sample Accuratea ff inity and kinetics data
Ligand
k a (1/Ms) 6.31E+05 7.13E+05 7.24E+05 6.23E+05 6.42E+05 5.61E+05 6.54E+05 8.75E+06 7.34E+05
k d (1/s) 1.51E-04 1.12E-03 8.46E-04 4.78E-04 2.63E-03 1.54E-04 2.85E-04 9.13E-04 1.31E-04
K D (M)
Di ff erentiate clones with similar a ff inity but di ff erent kinetics Strong correlation between crude and purified samples
Parental Ab
2.39E-10
Z0 Z1 Z2 Z3 Z4 Z5 Z6 P1 p2
No or weak binding
Binding to integrin
Peptide with low molecule weight
1.57E-09 1.17E-09 7.67E-10 4.10E-09 2.75E-10 4.36E-10 1.04E-10 1.78E-10
Humanization variants
PTM variants
TCR mutants binding to pMHC in different affinity range
Case Study 5: Simultaneous Binding for Bispecific Antibody Structural diversity in the bispecific antibody (bsAb) field has been rapidly expanded, with various formats being designed and optimized. In addition to evaluating binding a ff inity, simultaneous bind- ing is a critical characteristics for bsAbs.
Fig 3. Target Protein Binding Before and A ft er Drug Conjugation
Sample
k a (1/Ms) 1.25E+05 1.30E+05
k d (1/s) 1.73E-04 1.69E-04
K D (M)
Naked mAb
1.39E-09 1.30E-09
Fig 7. Simultaneous Binding by SPR
ADC
Sample 1 with high a ff inity to Ag2
Sample 2 with low a ff inity to Ag2
Step1
Step3
Step2
Ag1
BsAb
Bu ff er
Ag1
BsAb
Ag2
Case Study 2: Fc Receptor Characterization
Step3
Step2
Step2
Step3
Fig 4. SPR Binding Assay to Characterize FcRn and ADCC/ADCP/CDC E ff ector Function
K D detection in the presence of Ag1
BsAb
Ag1
Ag2
CM5chip
Conclusions WuXi Biologics has provided in-depth SPR characterization throughout drug discovery and development. High-throughput screening for lead identification from early discovery platform such as hybridoma and phage display. Lead ranking for optimization such as humanization and a ff inity maturation. Final lead characterization to evaluate binding a ff inity, epitope binding and e ff ector function. • • Acknowledgments We thank all scientists from WuXi Biologics’ CRO Services for their support during the method development. We also thank the legal department, public relations department. and marketing department for their help in patent filing and poster preparation and revision.
Fig 5. Fc E ff ector Characterization for Fc-Engineered Antibody
K D (M)
Fc engineered
Fc RIIa (R131) 4.31E-06
Fc RIIIa (F158) 3.95E-07
Fc RIIa (H131) 2.73E-06
Fc RIIIa (V158) 9.96E-08
Fc RI
Fc RIIb
FcRn at pH 6.0
Wild type hIgG1 Fc
7.85E-10
5.71E-06
3.74E-06
About WuXi Biologics CRO Services
8.47E-08 (~4-fold )
2.54E-08 (~4-fold )
3.37E-06
Afucosylated hIgG1 Fc
7.78E-10
2.64E-06
3.67E-06
4.69E-06
WuXi Biologics is a leading global CRDMO, with the R represented by our premium-quality CRO Services. With expertise in diverse drug modalities, we deliver comprehensive research services, including antibody discovery, protein production and engineering, and in vitro/in vivo testing, to seamlessly advance our clients’ molecules from concept to CMC development.
Fc RIIIa (F158) binding of WT (le ft )and afucosylatedAb(right)
Fc RIIIa (V158) binding of WT (le ft )and afucosylatedAb(right)
For more information see us at https://www.wuxibiologics.com/cro-services Drug Development Expertise Empowering Research Services for Biologics
SPR_Poster_202509_V1
To discuss this poster: PS_Marketing@wuxibiologics.com
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