Immobilization-Free Binding and Affinity Characterization of Higher Order Bispecific Antibody Complexes Using
Andreas V Madsen1, Oscar Mejias-Gomez1, Lasse E Pedersen1
1Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, Building 224, 2800 Kgs. Lyngby, Denmark.
Analytical Chemistry
|September 27, 2022
Summary
Flow-induced dispersion analysis (FIDA) characterizes bispecific antibodies (bsAbs) in solution, enabling analysis of binding interactions and cooperativity. This method offers a versatile tool for biotherapeutic development and manufacturing.
Area of Science:
- Biochemistry and Biophysics
- Protein Engineering
- Analytical Chemistry
Background:
- Bispecific antibodies (bsAbs) offer synergistic therapeutic potential by targeting multiple antigens simultaneously.
- Characterizing bsAbs is challenging due to their complex molecular architectures and binding interactions.
- Existing analytical methods often require surface immobilization, which can interfere with binding analysis.
Purpose of the Study:
- To introduce and validate flow-induced dispersion analysis (FIDA) for characterizing bsAbs.
- To demonstrate FIDA's ability to analyze bsAb binding interactions in solution under near-native conditions.
- To assess FIDA's utility for evaluating binding cooperativity and potential interference in bsAb-antigen interactions.
Main Methods:
- Utilized flow-induced dispersion analysis (FIDA) for the in-depth characterization of bsAbs.
- Determined individual equilibrium dissociation constants (Kd) in solution for bsAbs and their complexes with antigens.
- Analyzed binding cooperativity and potential interaction interference without surface immobilization.
Main Results:
- FIDA successfully characterized bsAbs with diverse molecular architectures and valencies in solution.
- Equilibrium dissociation constants were determined even for higher-order complexes involving both antigens.
- Binding functionality was illustrated by incremental increases in complex sizes upon exposure to antigens.
Conclusions:
- FIDA is a versatile tool for the comprehensive binding analysis of bsAbs.
- The method requires low material consumption and exhibits high matrix tolerability.
- FIDA is suitable for format selection and characterization of complex bi/multispecific protein therapeutics throughout development and manufacturing.
