sepharose and pyridine

A colorimetric method for determining cyanuric chloride (CC) and for monitoring its polysaccharide gel activation, before and after ligand binding, was developed. The method is based on the reaction of CC or its activated gel with pyridine and barbituric acid or dimethylbarbituric acid. The product formed yields a purple red color with λ max at 595 nm, and an E

Topics: Barbiturates; Color; Colorimetry; Pyridines; Sensitivity and Specificity; Sepharose; Solutions; Triazines; Water

The structure-based design and synthesis of four series of adsorbents for antibody purification by affinity chromatography has been investigated. The structures of 10 ligands were based on pyridine compounds that possessed thioalkyl substituents containing a primary amine, which was required for immobilisation of the ligands onto an epoxy-activated matrix (epoxy-Sepharose Fast Flow(®)). These new adsorbents were screened in monoclonal antibody binding assays in order to determine optimal buffer conditions for capture and elution under static and dynamic adsorption conditions. From batch binding measurements, the binding affinities, KD's, were found to be in the range of 3-5μM and the maximum capacities, qm's were between 12 and 30mgmAb/mL resin, depending on the substitution pattern of the thioalkylamine in the N-heterocyclic ring structure of the ligands. The amount of monoclonal antibody bound and eluted under overload conditions was influenced by the concentration of the sample loaded, the flow rate at which the sample was applied and the loading/volume. Further, the ability of these new adsorbents to selectively capture monoclonal antibodies of the class IgG1 from supernatants derived from genetically engineered CHO cells cultured in chemically defined media was investigated, documenting efficient capture and recovery of the mAb.

Topics: Adsorption; Animals; Antibodies, Monoclonal; CHO Cells; Chromatography, Affinity; Cricetinae; Cricetulus; Immunoglobulin G; Ligands; Pyridines; Sepharose

In this study, a pyridine-based compound, 4'-terpyridinylsulfanylethylamine (4'-TerPSEA), has been employed as a ligand to purify via mixed-mode chromatographic procedures a humanised monoclonal antibody of the IgG1 sub-class directly from crude supernatants derived from cultured CHO cells. The antibody binding capacity, selectivity and reusability of the adsorbent, derived from the immobilisation of this ligand onto Sepharose FF™, were compared to a Protein A affinity resin. The chromatographic performance of this mixed mode adsorbent was similar to that shown by the Protein A-based adsorbent with this IgG1 mAb. In addition, the IgG1 mAb was able to bind to the immobilised 4'-TerPSEA under reducing conditions. Through the use of papain-digested IgG1 mAb, fractionated with both the 4'-TerPSEA and Protein A adsorbents, it was found that this IgG1 mAb preferentially bound to the immobilised 4'-TerPSEA Sepharose FF™ resin through its Fc region.

Topics: 2,2'-Dipyridyl; Adsorption; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; CHO Cells; Chromatography, Liquid; Cricetinae; Cricetulus; Cysteamine; Immunoglobulin G; Ligands; Protein Binding; Pyridines; Resins, Synthetic; Sepharose; Staphylococcal Protein A