Profiling of oligosaccharide-mediated microheterogeneity of a monoclonal antibody by capillary electrophoresis.

Based on complex formation of borate with carbohydrates in alkaline solutions, the oligosaccharide microheterogeneity of a monoclonal antibody was studied using capillary zone electrophoresis. In borate buffers characteristic separation patterns were found that could be attributed to the same antibody by their UV spectra, while in a phosphate buffer, under otherwise the same conditions, only a single peak was observed. N- and O-glycans were chemically hydrolyzed by trifluoromethane sulfonic acid, resulting in a completely deglycosylated protein; alternatively, N-glycans were enzymatically cleaved by incubation with peptide N-glycosidase F (PNGase F).

Characterization of yeast cultivations by steric sedimentation field-flow fractionation.

The characterization and quantification of biomass is often time consuming and dependent on the cultivation media and gives no detailed information between cell size and shape and their productivity. By monitoring the bioprocess with steric sedimentation field-flow fractionation (Sd/StFFF) in combination with laser light scattering, not only cell growth, but also the variation of cell size and shape during the cultivation, can be observed. In this work, the feasibility of separating and characterizing cell populations by steric sedimentation field-flow fractionation is demonstrated by its application to three different yeast cultivation broths.

Analysis of synthetic peptides using matrix-assisted laser desorption ionization mass spectrometry.

Matrix-assisted laser desorption ionization mass spectroscopy (LDI MS), a novel method for analysis of large molecules, has been used for characterization of synthetic peptides and their by-products. The potential of LDI MS is demonstrated by analyzing crude synthetic peptides representing typical members of newly designed peptides and proteins. In the first case, a fragment condensation reaction yielding a highly hydrophobic six-helic bundle template-assembled synthetic protein (TASP) is monitored.

Free-flow electrophoresis for the purification of proteins: II. Isoelectric focusing and field step electrophoresis.

Two modes of continuous isoelectric focusing are described. The development of a natural pH gradient, consisting of a mixture of three buffer solutions, and the focusing behavior of human serum albumin is investigated. The advantages of isoelectric focusing in an artificial pH gradient of three buffer solutions are demonstrated on the purification of alpha-amylase from an E.

Scale-up of free-flow electrophoresis: II. Purification of alcohol dehydrogenase from a crude yeast extract by field step electrophoresis and combined field step-zone electrophoresis.

Results of the purification of alcohol dehydrogenase (ADH) by field step electrophoresis and combined field step-zone electrophoresis are presented. In field step electrophoresis, optimization of voltage, residence time and pH of the sample solution led to a maximal purification factor of 2.8 and a yield of 89% ADH.