Stable Dried Catalase Particles Prepared by Electrospraying

Abstract

Therapeutic proteins and peptides are clinically important, offering potency while reducing the potential for off-target effects. Research interest in developing therapeutic polypeptides has grown significantly during the last four decades. However, despite the growing research effort, maintaining the stability of polypeptides throughout their life cycle remains a challenge. Electrohydrodynamic (EHD) techniques have been widely explored for encapsulation and delivery of many biopharmaceuticals. In this work, we explored monoaxial electrospraying for encapsulation of bovine liver catalase, investigating the effects of the different components of the electrospraying solution on the integrity and bioactivity of the enzyme. The catalase was successfully encapsulated within polymeric particles made of polyvinylpyrrolidone (PVP), dextran, and polysucrose. The polysorbate 20 content within the electrospraying solution (50 mM citrate buffer, pH 5.4) affected the catalase loading-increasing the polysorbate 20 concentration to 500 μg/mL resulted in full protein encapsulation but did not prevent loss in activity. The addition of ethanol (20% v/v) to a fully aqueous solution improves the electrospraying process by reducing surface tension, without loss of catalase activity. The polymer type was shown to have the greatest impact on preserving catalase activity within the electrosprayed particles. When PVP was the carrier there was no loss in activity compared with fresh aqueous solutions of catalase. The optimum particles were obtained from a 20% w/v PVP or 30% w/v PVP-trehalose (1:1 w/w) solution. The addition of trehalose confers stability advantages to the catalase particles. When trehalose-PVP particles were stored at 5 °C, enzymatic activity was maintained over 3 months, whereas for the PVP-only analogue a 50% reduction in activity was seen. This demonstrates that processing catalase by monoaxial electrospraying can, under optimised conditions, result in stable polymeric particles with no loss of activity.

Keywords: bovine liver catalase; electrospraying; protein stability; proteins; solid-state protein formulation.

Figure 1

Area under the curve of catalase solutions (50 μg/mL), determined by SEC after storage at different ethanol concentrations (a) peak 1, (b) peak 2.

Figure 2

Catalase activity after storage at pH 5.4 in the presence of polysorbate 20 or polysorbate 80 at three different surfactant-to-catalase ratios (a) at 50 μg/mL catalase for 6 h, (b) at 1 mg/mL catalase for 24 h.

Figure 3

Protein content and activity for the catalase-dextran (1 μg/mg) particles electrosprayed from solutions containing 0–500 μg/mL of polysorbate 20. (a) catalase content, (b) catalase activity.

Figure 4

Protein content and activity for catalase-polymer particles (1 μg/mg) containing three different polymers (10% w/v in solution): (a) catalase content, (b) catalase activity.

Figure 5

Protein content and activity in electrosprayed catalase-PVP (1 μg/mg) and catalase-PVP-trehalose (1 μg/mg) particles obtained from 10%, 15%, and 20% w/v PVP or 20% w/v and 30% PVP-trehalose (1:1) solutions.

Figure 6

Scanning electron microscopy images of the electrosprayed catalase-PVP (1 μg/mg) particles prepared with solutions containing (w/v) (a) 10% PVP, (b) 15% PVP, (c) 20% PVP, (d) 20% PVP-trehalose, and (e) 30% PVP-trehalose. The scale bar represents 10 μm.

Figure 7

Particle size distribution of the electrosprayed catalase-PVP particles prepared from solutions containing (w/v) (a) 10% PVP, (b) 15% PVP, (c) 20% PVP, (d) 20% PVP-trehalose, and (e) 30% PVP-trehalose.

Figure 8

FTIR spectra of the raw materials and protein-loaded particles prepared from PVP solutions, showing (a) full spectrum and (b) enlargement of the 1750–650 cm⁻¹ region, and PVP-trehalose solutions: (c) full spectrum and (d) enlargement of the 1750–650 cm⁻¹ region.

Figure 8

FTIR spectra of the raw materials and protein-loaded particles prepared from PVP solutions, showing (a) full spectrum and (b) enlargement of the 1750–650 cm⁻¹ region, and PVP-trehalose solutions: (c) full spectrum and (d) enlargement of the 1750–650 cm⁻¹ region.

Figure 9

XRD diffraction patterns of the formulations and raw materials. (a) PVP particles, (b) PVP-trehalose particles.

Figure 10

Residual activity based on measured protein content within the polymeric particles (1 μg/mg catalase:polymer) and unformulated catalase stored over 90 days at (a) 5 °C, (b) room temperature, and (c) 40 °C/75% RH. Where the 30% PVP-trehalose formulation is statistically significant different to either 20% PVP (dark blue asterisk) or unprocessed catalase (light blue asterisk) at a given time point, this has been indicated by (*) p < 0.05 and (**) p < 0.01.