Sintered Glass Monoliths as Supports for Affinity Columns

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.

High-Purity Corundum as Support for Affinity Extractions from Complex Samples

“…These proteins, originating from the bacteria Staphylococcus aureus and Streptococcus, selectively bind immunoglobulins (IgG) from different species. [26] Similar isolation of IgG was already performed by using polymer monoliths [27], sintered glass monoliths, both functionalized with protein A [28] and on alumina monoliths with protein G [29]. The present work shows the application of nonporous, functionalized corundum without the demand for a chromatography system and with nearly unlimited potential for scale-up at a very low cost.…”

Efficient Purification of Cowpea Chlorotic Mottle Virus by a Novel Peptide Aptamer

“…The raw cylinder is glued into a titanium tube with the outer dimensions of 35 mm x 12 mm and a wall thickness of 1 mm using silicon F liquid, obtainable from Weicon (13200310). A detailed description of the construction and manufacturing of the column and the column holder is shown in previous work [37]. In this work, the monolith length was increased from 15 mm to 35 mm.…”

“…An assembled column system consisting of the monolithic column [37] built in a 3Dprinted column holder was connected to a syringe pump (Standard Infusion Only Pump 11 Elite from Harvard Apparatus, USA), and several reagents and washing solutions were pumped through the column. If not indicated otherwise, a flow rate of 1 mL/min was used, and the reactions took place at room temperature.…”

“…For the preparation of the affinity column, sintered glass monoliths were used as the starting material for the immobilization of the CCMV-binding peptide. This material and the evaluation of its performance regarding affinity separations were previously published [37]. The detailed protocol for the treatment of the monolithic raw column, its assembly, and the cleaning of the glass surface was performed as described there.…”

Cocaine Detection by a Laser-Induced Immunofluorometric Biosensor

“…Raw affinity columns were manufactured and prepared as described elsewhere [48]. Briefly, the column was cleaned, silanized with diethoxy(3-glycidyloxypropyl)-methyl silane and coated as described in Table S3.…”