Abstract
A novel stationary phase for affinity separations is presented. This material is based on sintered borosilicate glass readily available as semi-finished filter plates with defined porosity and surface area. The material shows fast binding kinetics and excellent long-term stability under real application conditions due to lacking macropores and high mechanical rigidity. The glass surface can be easily modified with standard organosilane chemistry to immobilize selective binders or other molecules used for biointeraction. In this paper, the manufacturing of the columns and their respective column holders by 3D printing is shown in detail. The model system protein A/IgG was chosen as an example to examine the properties of such monolithic columns under realistic application conditions. Several specifications, such as (dynamic) IgG capacity, pressure stability, long-term performance, productivity, non-specific binding, and peak shape, are presented. It could be shown that due to the very high separation speed, 250 mg antibody per hour and column can be collected, which surpasses the productivity of most standard columns of the same size. The total IgG capacity of the shown columns is around 4 mg (5.5 mg/mL), which is sufficient for most tasks in research laboratories. The cycle time of an IgG separation can be less than 1 min. Due to the glass material’s excellent pressure resistance, these columns are compatible with standard HPLC systems. This is usually not the case with standard affinity columns, limited to manual use or application in low-pressure systems. The use of a standard HPLC system also improves the ability for automation, which enables the purification of hundreds of cell supernatants in one day. The sharp peak shape of the elution leads to an enrichment effect, which might increase the concentration of IgG by a factor of 3. The final concentration of IgG can be around 7.5 mg/mL without the need for an additional nanofiltration step. The purity of the IgG was > 95% in one step and nearly 99% with a second polishing run.
Highlights
Many different stationary phases or supports have been used for sample preparation and chromatographic applications
The use of a standard HPLC system improves the ability for automation, which enables the purification of hundreds of cell supernatants in one day
Their usefulness for affinity separations was acknowledged, and the main solid supports were discussed in a review [13]
Summary
Many different stationary phases or supports have been used for sample preparation and chromatographic applications. For affinity separations [1,2], crosslinked dextrans, agaroses [3], celluloses, silica materials, acrylic, and epoxide polymers [4], porous glass, and many others can be used, all with their pros and cons [5,6]. The emergence of monolithic materials was considered a major step for more advanced stationary phases [8,9,10,11,12]. Their usefulness for affinity separations was acknowledged, and the main solid supports were discussed in a review [13]. Some commercial products are available [14], they could not gain a large market share yet
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