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.

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Materials

Bovine serum albumin (BSA) (Purity ≥ 98%) (Mol. Wt. 66.4 kDa), (2-hydroxypropyl)-β-cyclodextrin (HPβCD), gelatin hydrolysate enzymatic (hydrolyzed gelatin), and benzyl benzoate were procured from Millipore Sigma (St. Louis, MO, USA). Sodium phosphate monobasic monohydrate and sodium phosphate dibasic heptahydrate were procured from VWR international (Radnor, PA, USA).

Chanakya D. Patil, Yijing Huang, Kinnari Santosh Arte, Rachana Sapkota, Tianyun Zhang, Reza Babakhani Galangashi, Pavlos P. Vlachos, Eric J. Munson, Qi (Tony) Zhou, Li (Lily) Qu, Impact of hydrolyzed gelatin and hydroxypropyl-β-cyclodextrin as polymeric excipients on physical stability and injectability of high-concentration protein suspensions, International Journal of Pharmaceutics, 2026, 127361, ISSN 0378-5173, https://doi.org/10.1016/j.ijpharm.2026.127361.