Ingenieurwissenschaften und zugeordnete Tätigkeiten
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- Protein A (6) (entfernen)
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The aim of this work was the preparation of a novel stationary monolithic phase for affinity chromatography and HPLC-applications. Therefore, we have chosen porous glass filters that are available with different pore sizes as raw materials to prepare monolithic columns. We purchased VitraPOR 4 (10-16 μm pore size) and VitraPOR 5 (1.0-1.6 μm pore size) monolithic glass filters. The physical properties of these glass filters were characterized. The surface area, pore size distribution and the porosity were determined using mercury intrusion porosimetry and BET. These glass filters only exhibit flow through pores and therefore show no bimodal pore size distribution in the mercury intrusion curves. Due to their low permeability, the applied filters that exhibit an inner diameter of 8.0 mm and a length of 15.0±0.1 mm could be operated at flow rates more than 10 ml/min. High flow rates are favorable for fast separation experiments.
Das Ziel dieser Arbeit war es, die Vernetzung (Crosslinking) zwischen Protein G und einem Antikörper so durchzuführen, dass es zu keiner Modifikation der Antigen-Bindungsstelle des Antikörpers kommt. Zudem wurde im Rahmen dieser Arbeit ein Immunoassay entwickelt, mithilfe dessen zwanzig verschiedene Crosslinker in Hinblick auf ihre Fähigkeit Protein G zu funktionalisieren, untersucht werden konnten. Es konnten fünf Reagenzien identifiziert werden, die eine Derivatisierung von Protein G sowie eine anschließende Immobilisierung des Antikörpers auf dem Protein ermöglichen. Des Weiteren wurde gezeigt, dass mithilfe dieses Verfahrens sowohl die Immunpräzipitation als auch die massenspektrometrische Identifizierung und Charakterisierung von Proteinkomplexen erleichtert und verbessert werden kann.
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 nano-filtration step. The purity of the IgG was > 95% in one step and nearly 99% with a second polishing run.
Preactivation crosslinking – An efficient method for the oriented immobilization of antibodies
(2019)
Crosslinking of proteins for their irreversible immobilization on surfaces is a proven and popular method. However, many protocols lead to random orientation and the formation of undefined or even inactive by-products. Most concepts to obtain a more targeted conjugation or immobilization requires the recombinant modification of at least one binding partner, which is often impractical or prohibitively expensive. Here a novel method is presented, which is based on the chemical preactivation of Protein A or G with selected conventional crosslinkers. In a second step, the antibody is added, which is subsequently crosslinked in the Fc part. This leads to an oriented and covalent immobilization of the immunoglobulin with a very high yield. Protocols for Protein A and Protein G with murine and human IgG are presented. This method may be useful for the preparation of columns for affinity chromatography, immunoprecipitation, antibodies conjugated to magnetic particles, permanent and oriented immobilization of antibodies in biosensor systems, microarrays, microtitration plates or any other system, where the loss of antibodies needs to be avoided, and maximum binding capacity is desired. This method is directly applicable even to antibodies in crude cell culture supernatants, raw sera or protein-stabilized antibody preparations without any purification nor enrichment of the IgG. This new method delivered much higher signals as a traditional method and, hence, seems to be preferable in many applications.
Preactivation Crosslinking - An Efficient Method for the Oriented Immobilization of Antibodies
(2019)
Crosslinking of proteins for their irreversible immobilization on surfaces is a proven and popular method. However, many protocols lead to random orientation and the formation of undefined or even inactive by-products. Most concepts to obtain a more targeted conjugation or immobilization requires the recombinant modification of at least one binding partner, which is often impractical or prohibitively expensive. Here a novel method is presented, which is based on the chemical preactivation of Protein A or G with selected conventional crosslinkers. In a second step, the antibody is added, which is subsequently crosslinked in the Fc part. This leads to an oriented and covalent immobilization of the immunoglobulin with a very high yield. Protocols for Protein A and Protein G with murine and human IgG are presented. This method may be useful for the preparation of columns for affinity chromatography, immunoprecipitation, antibodies conjugated to magnetic particles, permanent and oriented immobilization of antibodies in biosensor systems, microarrays, microtitration plates or any other system, where the loss of antibodies needs to be avoided, and maximum binding capacity is desired. This method is directly applicable even to antibodies in crude cell culture supernatants, raw sera or protein-stabilized antibody preparations without any purification nor enrichment of the IgG. This new method delivered much higher signals as a traditional method and, hence, seems to be preferable in many applications.