RESUMEN
In this study, we studied the chromatographic performance of this newly developed wide pore superficially porous particles (SPPs) with 3.5 µm particle size and 450 Å pore size, for the separation of proteins and Immunoglobulin G antibodies. We studied the selectivity of different phases (C4, SB-C18 and Diphenyl), the effect of temperature, column carryover and column chemical lifetime. We also compared our SPPs with other wide pore SPPs in similar particle sizes and sub 2 µ wide pore totally porous particles by van Deemter studies and gradient separations of proteins and immunoglobulin G antibodies. The results showed that the SPPs containing larger pore size gave better chromatographic performance.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Inmunoglobulina G/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Humanos , Inmunoglobulina G/análisis , Inmunoglobulina G/química , Tamaño de la Partícula , Porosidad , Proteínas Recombinantes/análisis , Proteínas Recombinantes/químicaRESUMEN
In recent years, superficially porous particles (SPPs) have drawn great interest because of their special particle characteristics and improvement in separation efficiency. Superficially porous particles are currently manufactured by adding silica nanoparticles onto solid cores using either a multistep multilayer process or one-step coacervation process. The pore size is mainly controlled by the size of the silica nanoparticles and the tortuous pore channel geometry is determined by how those nanoparticles randomly aggregate. Such tortuous pore structure is also similar to that of all totally porous particles used in HPLC today. In this article, we report on the development of a next generation superficially porous particle with a unique pore structure that includes a thinner shell thickness and ordered pore channels oriented normal to the particle surface. The method of making the new superficially porous particles is a process called pseudomorphic transformation (PMT), which is a form of micelle templating. Porosity is no longer controlled by randomly aggregated nanoparticles but rather by micelles that have an ordered liquid crystal structure. The new particle possesses many advantages such as a narrower particle size distribution, thinner porous layer with high surface area and, most importantly, highly ordered, non-tortuous pore channels oriented normal to the particle surface. This PMT process has been applied to make 1.8-5.1µm SPPs with pore size controlled around 75Å and surface area around 100m(2)/g. All particles with different sizes show the same unique pore structure with tunable pore size and shell thickness. The impact of the novel pore structure on the performance of these particles is characterized by measuring van Deemter curves and constructing kinetic plots. Reduced plate heights as low as 1.0 have been achieved on conventional LC instruments. This indicates higher efficiency of such particles compared to conventional totally porous and superficially porous particles.
Asunto(s)
Técnicas de Química Analítica/métodos , Micelas , Dióxido de Silicio/síntesis química , Cromatografía Líquida de Alta Presión , Cinética , Tamaño de la Partícula , Porosidad , Reproducibilidad de los Resultados , Dióxido de Silicio/química , Dióxido de Silicio/normasRESUMEN
Superficially porous particles (SPPs) with pore size ranging from 90Å to 120Å have been a great success for the fast separation of small molecules over totally porous particles in recent years. However, for the separation of large biomolecules such as proteins, particles with large pore size (e.g. ≥ 300Å) are needed to allow unrestricted diffusion inside the pores. One early example is the commercial wide pore (300Å) SPPs in 5µm size introduced in 2001. More recently, wide pore SPPs (200Å and 400Å) in smaller particle sizes (3.5-3.6µm) have been developed to meet the need of increasing interest in doing faster analysis of larger therapeutic molecules by biopharmaceutical companies. Those SSPs in the market are mostly synthesized by the laborious layer-by-layer (LBL) method. A one step coating approach would be highly advantageous, offering potential benefits on process time, easier quality control, materials cost, and process simplicity for facile scale-up. A unique one-step coating process for the synthesis of SPPs called the "coacervation method" was developed by Chen and Wei as an improved and optimized process, and has been successfully applied to synthesis of a commercial product, Poroshell 120 particles, for small molecule separation. In this report, we would like to report on the most recent development of the one step coating coacervation method for the synthesis of a series of wide pore SPPs of different particle size, pore size, and shell thickness. The one step coating coacervation method was proven to be a universal method to synthesize SPPs of any particle size and pore size. The effects of pore size (300Å vs. 450Å), shell thickness (0.25µm vs. 0.50µm), and particle size (2.7µm and 3.5µm) on the separation of large proteins, intact and fragmented monoclonal antibodies (mAbs) were studied. Van Deemter studies using proteins were also conducted to compare the mass transfer properties of these particles. It was found that the larger pore size actually had more impact on the performance of mAbs than particle size and shell thickness. The SPPs with larger 3.5µm particle size and larger 450Å pore size showed the best resolution of mAbs and the lowest back pressure. To the best of our knowledge, this is the largest pore size made on SPPs. These results led to the optimal particle design with a particle size of 3.5µm, a thin shell of 0.25µm and a larger pore size of 450Å.