(T1430-03-13) Optimizing Protein Encapsulation Condition in Cholesterol-Modified Hyaluronic Acid (CHHA) Nanogels for Sustained-Release of APIs

Purpose: We have been developing CHHA as a new pharmaceutical excipient and establishing the industrial manufacturing process. Prior, we reported that CHHA formed nanogel in water, and encapsulated APIs like small molecules, peptides, and proteins by just mixing (Figure 1). In pursuit of applying nanogels to the rapidly growing field of biopharmaceuticals, we explored the optimal conditions for protein encapsulation and the potential as a sustained-release formulation.

Methods: CHHA and human growth hormone (hGH, pI~5) were dissolved in water in different vials. The hGH solution was added to the CHHA solution prepared with a specific pH between 5.5 and 11, or salt concentration between 0 mM to 150 mM (CHHA: hGH = 100: 50, w/w). After incubation for 24 hours between 4℃ and 75℃, CHHA-hGH solution was diluted by 2xPBS and centrifuged (x9000g, 5min, 4℃). The supernatant was recovered and used it as the size exclusion chromatography (SEC) sample. The non-encapsulated hGH was quantified by SEC. Encapsulation ratio was calculated using the following equation: [encapsulated hGH] = [prepared hGH] – [non-encapsulated hGH] (Encapsulation ratio) = [encapsulated hGH]/ [prepared CHHA]. In the pharmacokinetics test, the CHHA-hGH formulation was prepared using the method described earlier. After equilibration and sterile filtration through a 0.2μm filter, the CHHA-hGH formulation was subcutaneously injected into SD rats. Plasma samples were collected at various time points, and the hGH concentration in plasma was measured by ELISA.

Results: We confirmed the influences of encapsulation conditions such as the pH, the salt concentration, and the incubation temperature. As for the pH, the increase in the encapsulation ratio was observed as the pH approached the isoelectric point and the highest encapsulation ratio (47wt%) was achieved at the pH proximity to the isoelectric point. Regarding the salt concentration, the low salt concentration tended to increase the encapsulation ratio. In relation to the temperature, the higher temperature tended to increase the encapsulation ratio. In the pharmacokinetics test, we detected hGH in plasma for 11days by using CHHA-hGH formulation while hGH without CHHA disappeared less than a day (Figure 2).

Conclusion: We found the optimal condition of preparing CHHA-protein formulations by adjusting the pH, the salt concentration, and the incubation temperature. Controlling these factors would be important to regulate the interactions between CHHA nanogels and proteins, and to efficiently achieve formulation preparation. According to the pharmacokinetics results, we could expect that CHHA nanogel formulation would be used as a few weeks sustained-release drug carrier. As the future tasks, we will make effort to elucidate the location of APIs in nanogel and extend release period.

References: T. Nakai, et al. Macromolecular Bioscience. 2012. 475-483.
Thore W. Wilhelmsen, et al. Procedia Chemistry, 2010. 34-45.

Acknowledgements: All animal experiments were conducted under approval from the Animal Care Committee of "each CRO". These experiments were carried out in compliance with the Act on Welfare and Management of Animals and related guidelines.


Figure 1. Schematic illustration of cholesterol-modified hyaluronic acid and encapsulation APIs.


Figure 2. Plasma pharmacokinetic of human Growth Hormone (hGH) after subcutaneously injected into SD rats.