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

Liquid-liquid phase separation (LLPS) of protein solutions has been usually related to strong protein-protein interactions (PPI) under certain conditions. For the first time, we observed the LLPS phenomenon for a novel protein modality, peptide-fused monoclonal antibody (pmAb). LLPS emerged within hours between pH 6.0 to 7.0 and disappeared when solution pH values decreased to pH 5.0 or lower. Negative values of interaction parameter (kD) and close to zero values of zeta potential (ζ) were correlated to LLPS appearance. However, between pH 6.0 to 7.0, a strong electrostatic repulsion force was expected to potentially avoid LLPS based on the sequence predicted pI value, 8.35. Surprisingly, this is significantly away from experimentally determined pI, 6.25, which readily attributes the LLPS appearances of pmAb to the attenuated electrostatic repulsion force. Such discrepancy between experiment and prediction reminds the necessity of actual measurement for a complicated modality like pmAb. Furthermore, significant protein degradation took place upon thermal stress at pH 5.0 or lower. Therefore, the effects of pH and selected excipients on the thermal stability of pmAb were further assessed. A formulation consisting of arginine at pH 6.5 successfully prevented the appearance of LLPS and enhanced its thermal stability at 40 °C for pmAb. In conclusion, we have reported LLPS for a pmAb and successfully resolved the issue by optimizing formulation with aids from PPI characterization.

Long-acting MIC-1/GDF15 molecules to treat obesity: Evidence from mice to monkeys.

In search of metabolically regulated secreted proteins, we conducted a microarray study comparing gene expression in major metabolic tissues of fed and fasted ob/ob mice and C57BL/6 mice. The array used in this study included probes for ~4000 genes annotated as potential secreted proteins. Circulating macrophage inhibitory cytokine 1 (MIC-1)/growth differentiation factor 15 (GDF15) concentrations were increased in obese mice, rats, and humans in comparison to age-matched lean controls. Adeno-associated virus-mediated overexpression of GDF15 and recombinant GDF15 treatments reduced food intake and body weight and improved metabolic profiles in various metabolic disease models in mice, rats, and obese cynomolgus monkeys. Analysis of the GDF15 crystal structure suggested that the protein is not suitable for conventional Fc fusion at the carboxyl terminus of the protein. Thus, we used a structure-guided approach to design and successfully generate several Fc fusion molecules with extended half-life and potent efficacy. Furthermore, we discovered that GDF15 delayed gastric emptying, changed food preference, and activated area postrema neurons, confirming a role for GDF15 in the gut-brain axis responsible for the regulation of body energy intake. Our work provides evidence that GDF15 Fc fusion proteins could be potential therapeutic agents for the treatment of obesity and related comorbidities.