Investigating Weak Polypeptide-Cyclodextrin Interactions in Biologic Formulation Development Using Affinity Capillary Electrophoresis and Flow-Induced Dispersion Analysis.

Abstract

Understanding protein-excipient interactions is vital for biopharmaceutical formulation, as they influence stability and pharmacokinetics (PK). Cyclodextrins (CDs) are widely used excipients that enhance solubility and stability, but their weak interactions with polypeptides remain poorly characterized. Relaxin (RLX), a potent anti-heart failure polypeptide, was selected due to its PK relevance and in vivo interaction with human serum albumin (HSA). Given RLX's poor solubility, CDs were identified as the most effective solubilizers. However, traditional affinity assays lack the sensitivity to detect weak CD-polypeptide interactions. To overcome this limitation, we employed affinity capillary electrophoresis and flow-induced dispersion analysis (FIDA) to assess RLX's binding with hydroxypropyl-β-cyclodextrin (HP-β-CD) and sulfobutylether-β-cyclodextrin (SBE-β-CD). Our results showed a higher affinity for SBE-β-CD than HP-β-CD, though both interactions were significantly weaker than RLX's binding to HSA. These findings provide key insights into weak CD-polypeptide interactions, supporting SBE-β-CD as an excipient to improve solubility without compromising PK performance. Additionally, the effectiveness of these rapid, nonconventional analytical methods was validated through in vivo PK studies in a cynomolgus monkey model, highlighting their value in excipient-protein binding research.