Resolving Liquid-Liquid Phase Separation for a Peptide Fused Monoclonal Antibody by Formulation Optimization.

Area of Science:

• Biochemistry
• Protein Chemistry
• Formulation Science

Background:

• Liquid-liquid phase separation (LLPS) in protein solutions is typically linked to strong protein-protein interactions (PPI).
• Peptide-fused monoclonal antibodies (pmAbs) represent a novel protein modality with unique characteristics.

Purpose of the Study:

• To investigate the phenomenon of LLPS in pmAbs.
• To understand the factors influencing LLPS and thermal stability of pmAbs.
• To develop a formulation strategy to mitigate LLPS and enhance stability.

Main Methods:

• Observation of LLPS under varying pH conditions.
• Measurement of interaction parameter (k_D) and zeta potential (ζ).
• Assessment of protein degradation under thermal stress.
• Formulation development using arginine and pH optimization.

Main Results:

• LLPS was observed in pmAbs between pH 6.0-7.0, correlating with negative k_D and near-zero ζ.
• Experimentally determined pI (6.25) differed significantly from predicted pI (8.35), explaining attenuated electrostatic repulsion and LLPS.
• Protein degradation occurred at pH ≤ 5.0 under thermal stress.
• A formulation with arginine at pH 6.5 prevented LLPS and enhanced thermal stability at 40°C.

Conclusions:

• LLPS is a phenomenon that can occur in pmAbs.
• Accurate experimental determination of properties like pI is crucial for complex modalities.
• Formulation optimization, including pH and excipients like arginine, can effectively manage LLPS and improve pmAb stability.