Selection of bispecific antibodies with optimal developability using FcRn‑Ph‑HPLC as an optimized FcRn affinity...

Area of Science:

• Biochemistry
• Immunology
• Pharmacology

Background:

• Predicting therapeutic antibody pharmacokinetics (PK) early in development is crucial.
• Neonatal Fc receptor (FcRn) binding efficiency significantly influences immunoglobulin (IgG) serum half-life.
• Existing FcRn high-performance liquid chromatography (HPLC) assays require modification for physiological context and accurate half-life prediction.

Purpose of the Study:

• To present a modified FcRn-HPLC assay, termed FcRn-pH-HPLC, for reproducible measurement of FcRn dissociation pH.
• To correlate FcRn dissociation pH with established in vivo half-lives of therapeutic antibodies.
• To evaluate the impact of antibody modifications and bispecific formats on FcRn dissociation pH.

Main Methods:

• Development and application of the FcRn-pH-HPLC assay.
• Measurement of FcRn dissociation pH for various IgG-based antibodies and bispecifics from the ROCK® platform.
• Correlation analysis between measured FcRn dissociation pH and known in vivo antibody half-lives.

Main Results:

• The FcRn-pH-HPLC method reproducibly measures FcRn dissociation pH, enabling correlation with in vivo half-lives.
• Antibody format, including target and effector antigen-binding domain presentation and orientation in bispecifics, influences FcRn retention.
• Fc domain modifications and stabilizing disulfide bonds showed minimal impact on FcRn dissociation pH.

Conclusions:

• FcRn-pH-HPLC provides a valuable tool for interpreting FcRn binding in a physiological context, improving in vivo half-life estimations.
• Understanding how antibody design impacts FcRn binding facilitates the development of therapeutic antibodies with tailored half-lives.
• This method can inform the design of mono-, bi-, and multi-specific antibodies with optimized developability.