Computational fluid dynamics (CFD) insights into agitation stress methods in biopharmaceutical development.

Agitation of small amounts of liquid is performed routinely in biopharmaceutical process, formulation, and packaging development. Protein degradation commonly results from agitation, but the specific stress responsible or degradation mechanism is usually not well understood. Characterization of the agitation stress methods is critical to identifying protein degradation mechanisms or specific sensitivities. In this study, computational fluid dynamics (CFD) was used to model agitation of 1 mL of fluid by four types of common laboratory agitation instruments, including a rotator, orbital shaker, magnetic stirrer and vortex mixer. Fluid stresses in the bulk liquid and near interfaces were identified, quantified and compared. The vortex mixer provides the most intense stresses overall, while the stir bar system presented locally intense shear proximal to the hydrophobic stir bar surface. The rotator provides gentler fluid stresses, but the air-water interfacial area and surface stresses are relatively high given its low rotational frequency. The orbital shaker provides intermediate-level stresses but with the advantage of a large stable platform for consistent vial-to-vial homogeneity. Selection of experimental agitation methods with targeted types and intensities of stresses can facilitate better understanding of protein degradation mechanisms and predictability for "real world" applications.

Uniform encapsulation of stable protein nanoparticles produced by spray freezing for the reduction of burst release.

Stable protein nanostructured particles, produced by spray freezing into liquid (SFL) nitrogen, were encapsulated uniformly into microspheres to reduce the burst release over the first 24 h. The denaturation and aggregation of these bovine serum albumin (BSA) high-surface area particles were minimal due to ultra-rapid freezing and the absence of a liquid-air interface. Upon sonication, these friable highly porous, solid protein particle aggregates broke up into submicron particles. These particles were encapsulated into DL-lactide/glycolide copolymer (PLGA) and poly(lactic acid) (PLA) microspheres by anhydrous solid-in-oil-in-oil (s/o/o) techniques. For 5% loading of protein, the burst release after 24 h was only 2.5-4.1%, that is, values fivefold to tenfold lower than those observed for larger more conventional BSA particles. At a loading of 10%, the burst was only 6 and 13% for PLGA and PLA, respectively, and at 15% loading it was only 12% for PLGA. As shown with confocal and scanning electron microscopy (SEM), the low burst is consistent with a uniform distribution of protein nanoparticles, which were about 100 times smaller than the microspheres. Changes in aggregation and secondary structure, which were monitored by size exclusion chromatography and FTIR, respectively, indicated only slight monomer loss (3.9%) and high structural integrity (38% alpha-helix) in the encapsulated protein.

Encapsulation of protein nanoparticles into uniform-sized microspheres formed in a spinning oil film.

A new spinning oil film (SOF) solid-in-oil-in-oil emulsion process was developed to produce uniform-sized protein-loaded biodegradable microspheres. A thin SOF on a cylindrical rotor was used to shear droplets from a nozzle tip to control droplet size. The resulting microspheres with low polydispersity (6%) produced a low burst (6%-11%) release even at high loadings (13%-18% encapsulated solids, 8%-12% protein). The SOF process had a high yield and did not require the presence of water, which can cause protein denaturation, or surfactants, which may be unwanted in the final product. Amorphous protein and crystalline excipient solids were encapsulated into 3 different polymers, giving a homogenous drug distribution throughout the microspheres, and an essentially complete protein encapsulation efficiency (average = 99%). In contrast, large burst release was observed for polydisperse microspheres produced by a conventional emulsification technique, particularly for microspheres smaller than 25 mum in diameter, which gave 93% burst at 15% loading. The uniform encapsulation of high loadings of proteins into microspheres with low polydispersity in an anhydrous process is of practical interest in the development of controlled-release protein therapeutics.