Fast isolation of proteins from bioreactors using affinity chromatography techniques

Abstract: The purification of proteins from raw materials like fermentation broth and other biological sources usually involves several purification steps and is rather time consuming and expensive. This is a major concern in the production of many important protein products e.g. pharmaceuticals such as insulin and blood coagulation factors. One way to speed up the isolation of protein from particle-containing liquids is by the use of expanded bed adsorption. By a selective adsorption of the desired protein, it can be clarified from particles, concentrated and purified from many molecular impurities in one step. A new improved support material for the use in expanded bed adsorption was developed and is described in this thesis. It was shown to have excellent adsorption efficiency at flow rates higher than normally used today. The new, pelicular, matrix was used to capture antibodies from cell-containing fermentation broth. When particle-free samples are to be purified, a packed bed can be used. In an industrially relevant application, the isolation of factor VIII from clarified fermentation broth, the recently developed matrix material “superporous agarose” was employed. Compared to traditional agarose supports it showed considerably higher efficiency at increased flow rates. This was ascribed to the presence of superpores that allow intraparticle flow, which reduces the diffusion distances. In analyses of cell-containing samples small columns permeable to cells and cell debris are valuable tools. A miniaturised expanded bed column and a small superporous monolithic column were designed and used in experimental set-ups in which cell containing samples were analysed for e.g. IgG and glucose. When new support materials are developed it is important to be able to compare their performance with existing supports. The methods used in determining the efficiency of expanded beds were critically examined and found unsuitable when comparing beds of different degrees of expansion. Quantities specially modified for expanded bed evaluation were therefore developed.