Impact of hydrolyzed gelatin and hydroxypropyl-β-cyclodextrin as polymeric excipients on physical stability and injectability of high-concentration protein suspensions.

Abstract

Protein-based therapeutics at high-concentrations often face significant development challenges, including high viscosity, limited solubility, poor injectability, and instability concerns. Non-aqueous protein suspensions offer a promising strategy to achieve high protein concentrations while maintaining acceptable viscosity and injectability. In this study, we evaluated the impact of two polymeric excipients (hydrolyzed gelatin, hydroxypropyl-β-cyclodextrin or HPβCD) and their combinations on the viscosity, injectability, and stability of high-concentration non-aqueous suspensions containing bovine serum albumin (BSA). Suspension viscosity was characterized by rheological measurement, while injectability was assessed using a custom-built setup to measure plunger force through a syringe with a 27G needle. Spray-dried powders and the corresponding suspensions were subjected to accelerated physical stability (monomer loss) studies. Protein stability and structural integrity were evaluated using size-exclusion chromatography (SEC), circular dichroism (CD), and solid-state NMR (ssNMR), while X-ray photoelectron spectroscopy (XPS) was used to assess surface chemical properties of spray-dried particles. Hydrolyzed gelatin provided approximately five-fold greater preservation of monomer content during storage, indicating enhanced protein stability (∼1.5% monomer loss in 90-day stressed storage). In contrast, HPβCD significantly improved injectability, reducing injection force by approximately 5 N compared with formulations containing protein alone (13 N to 8 N). The combination of hydrolyzed gelatin and HPβCD yielded stable and injectable suspensions with protein loadings of 150 - 250 mg/mL. The current work highlighted the potential of polymer-based excipient systems for developing high-concentration injectable suspensions of proteins.