Affinity chromatography, large

Sepharose (e.g. Sepharose CL and Bio-Gel A) is a bead form of agarose gel which is useful for the fractionation of high molecular weight substances, for molecular weight determinations of large molecules (molecular weight > 5000), and for the immobilisation of enzymes, antibodies, hormones and receptors usually for affinity chromatography applications. 

To allow all culture productiou to be coutrolled, a method for rapid analysis is required. Prior to development of an LC-MS method, the analysis was both complex and time-consuming, involving the purification of a relatively large amount of the antibody using affinity chromatography, enzymatic release, and subsequent derivatizafion of the oligosaccharides and their analysis by using capillary electrophoresis. 

The endothelin B receptor is an example of characterization of a homogeneous, affinity purified protein (Roos et al., 1998). Significant progress has been made in the development of techniques for more high-throughput identification of phosphorlyation events. Analysis of large sets of phosphorylated proteins is facilitated by the availability of affinity purification methods such as anti-phosphotyrosine or anti-phosphoserine antibodies or metal affinity chromatography (Neubauer and Mann, 1999 Soskic et al., 1999). These methods are not specific to a particular protein but rather are used to fractionate all proteins that are phosphorylated. 

Bis-hydrazide-containing molecules also can be used to activate soluble polymeric sub-stances-containing aldehyde groups. For instance, dextran may be periodate oxidized to create numerous formyl functionalities on each molecule. Subsequent reaction with a homobifunctional hydrazide in large excess results in a hydrazide-activated polymer having multivalent-binding capability toward aldehydes or ketones (Chapter 25, Section 2.2). Insoluble support matrices suitable for affinity chromatography have been activated in a similar fashion to create the hydrazide derivative (O Shannessy and Wilchek, 1990). 

MS can perform large-scale analyses of protein interactions. Interacting partners of proteins in complexes can be purified by several strategies including affinity chromatography and immunoprecipi-tation with antibodies specific to the bait protein. The purified components can then be subjected to LC-MS/MS and proteins within the complex identified. Using a variety of approaches including... 

The first step in biopharmaceutical development is the selection of a clone in a specific cell line. Whole-mass analysis, if possible, is a fairly simple and powerful tool at this stage to verify the successful expression and translation of the desired protein. VanAdrichem et al.65 described the use of MALDI MS to monitor protein expression in several mammalian cell lines like CHO DXB11, CHO SSF3, and hybridomas. Quantitative MALDI-TOF MS measurements of an IgG antibody and insulin during large-scale production in hybridoma cells were comparable to affinity chromatography results. 

The ability of proteins to form enantioselective interactions with a large variety of drugs is used in chiral affinity chromatography. Protein CSPs that are most frequently used for the enantioseparation of pharmaceuticals include bovine serum albumin (BSA), human serum albumin... 

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