Purpose: CSP7 is a domain of caveolin-1 scaffolding peptide and has been reported as a nebulized dosage form for the treatment of idiopathic pulmonary fibrosis. Air-jet milling (AJM) is a particle size reduction process that uses high velocity jets of gas, such as nitrogen, to impart energy to particles, causing inter-particle collisions and attrition [2, 3]. Applications of air jet milling for particle size reduction of solid forms of biopharmaceuticals, especially excipient free formulations, are hindered by concerns that the shear stress from the milling process can affect the structural integrity of the macromolecules resulting in loss of biological activity[4-6].
Methods: Bulk CSP7 drug substance (lyophilized) was purchased from Polypeptide Laboratories (San Diego, CA, USA), and then air-jet milled using a Model 00 Jet-O-Mizer™ (Telford, PA, USA). Briefly, CSP7 powder was fed into the grinding chamber at a rate of 1 g/min, with fixed push and grinding pressure. After one cycle, the powder was retrieved from different parts of the mill in the following fractions: collection bag + bag adaptor, tube after grinding chamber + grinding chamber (bfC), cyclone, collection vessel adapter and collection vessel. All of the fractions were mixed using TURBULA® Shaker-Mixer (Glen Mills, New Jersey, USA).
Geometric Particle Size Distribution (GPSD) of AJM CSP7 was determined using Symptec Laser Diffraction instrument equipped with solid attachment. Primary pressure and feeding rate were 3 bars and 20% rotation, respectively. Aerodynamic Particle Size Distribution of AJM CSP7 was examined using Next Generation Impactor (NGI, MSP Corp., Shoreview, MN). One capsule was run, and each sample was run in triplicate. Data was analyzed with Copley Inhaler Testing Data Analysis Software (Copley Scientific, Nottingham UK).
Chemical potency after air-jet milling and stability after storage in different conditions, including accelerated condition, was evaluated using High Performance Liquid Chromatography (HPLC). Unprocessed and AJM CSP7 samples were stored in three conditions: 4 °C, 25 °C/60 relative humidity (RH), and 40 °C/75RH, respectively. At each condition, samples are exposed to or isolated from the environment by uncapping or capping the containers of powders. Potencies are tested at Day 0, 5, 15 and 32.
Results: Geometric particle size distribution from each fraction of milled sample was measured and analyzed. In Table 1, Dv (10), Dv (50), and Dv (90) are compared among unprocessed CSP7, milled particles from each part of air-jet miller, and the final mixture of AJM CSP7. AJM decreased the particle size from Dv (50) 5.3 to 2.1 µm, and the fine particle fraction (1-5 µm) ranged from 37.5% to 75.4%. Particles from all fractions of the AJM process are thus usable, which enhanced the final yield.
Table 1. Geometric particle size distribution of AJM and unprocessed CSP7
Dv 10 (µm) Dv 50 (µm) Dv 90 (µm) % 1-5 µm
Unprocessed 1.4±0.0 5.3±0.0 22.0±1.7 37.5±1.8
Bag+bag adaptor 0.7±0.0 1.5±0.0 3.3±0.1 68.4±1.0
bfc+chamber 1.1±0.1 2.8±0.2 5.4±0.2 78.6±1.3
cyclone 0.9±0.0 2.5±0.2 5.0±0.2 78.0±1.1
vessel adaptor 1.0±0.0 2.4±0.0 4.7±0.1 82.2±0.8
collection vessel 1.0±0.0 2.5±0.0 4.9±0.1 80.6±0.6
Mix 0.8±0.0 2.1±0.0 4.6±0.0 75.4±0.7
Aerodynamic particle size distribution was tested using NGI. Data in Table 2 show that 66.5% particles become fine particles after milling, resulting in up to approximately 83% particles are inhalable CSP7. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) are also reduced from 3.0 to 2.2 µm and 2.5 to 1.9 µm, respectively. Moreover, the deposition of CSP7 in each stage of the NGI is shown in Figure 1. About 70% of unprocessed CSP7 particles are trapped in the throat and pre-separator, resulting in fewer particles reaching later stages of the NGI. In contrast, the majority of AJM CSP7 particles deposit in stages 2 to 6, which are ideal stages for inhalable particles.
Table 2. Fine Particle Fraction (FPF), Mass Median Aerodynamic Diameter (MMAD), and Geometric Standard Deviation (GSD) of unprocessed and air-jet milled CSP7
FPF% (<5 µm) MMAD (µm) GSD (µm)
Unprocessed CSP7 16.4 ± 0.9 3.0 ± 0.3 2.5 ± 0.0
Air Jet Milled CSP7 82.9 ± 5.7 2.2 ± 0.0 1.9 ± 0.0
The stability of AJM CSP7 is evaluated by determining the chemical potency by HPLC (Figure 2). No significant potency loss was observed after 32 days storage at the three stability conditions.
Conclusion: Air-jet milling was successfully used to reduce particle size of therapeutic peptide CSP7 to produce excipient free inhalable dry powder without compromising its chemical potency. Geometric and aerodynamic particle size distributions demonstrate that the micronized powder was suitable for lung delivery. The study enables development of CSP7 dry powder inhaler for the treatment of idiopathic pulmonary fibrosis.