The affinity precipitation for the isolation of biomolecules

Product isolation increasingly becomes a major bottleneck in molecular biotechnology. This is especially the case for the sensitive proteineous substances (recombinant proteins, antibodies) and lately also DNA, usually produced in low concentration. The current challenge to preparative bioseparation can be summed up as the need to perform an economically sound, high-resolution separation at large scale, while maintaining "physiological" conditions throughout. Currently the only approach, which allows the direct, selective enrichment of the product from a highly diluted and complex feed, is to use biospecific interactions, i.e. "affinity" techniques. Affinity chromatography, the best known and understood of these methods may not be ideally suited early on during the downstream process because of danger of column fouling with most raw feeds, but also because of limited scale up potential and high costs (equipment, material). A promising technique combining the affinity interaction and the precipitation technique, called affinity precipitation, significantly increases the selectivity while retaining the main advantages of the precipitation method like the relatively simple equipment requirements and the applicability at large industrial scale. Affinity precipitation should also have advantages in terms of scalability, handling, and costs. In affinity precipitation, the ability of certain water-soluble polymers to form a separate phase under particular environmental conditions is used to isolate and purify biomolecules. The precipitation requires only small changes in the environment (pH, ionic strength, temperature, light, presence of specific substances). Such polymers are often called "stimuli-responsive" polymers. PolyNIPAAm is one of the most studied thermoresponsive polymers. Such molecules typically show a critical solution temperature (CST) in aqueous solution, i.e. they are soluble in cold water, but become insoluble and precipitate once the CST is surpassed. The phenomenon is usually fully reversible and the molecules redissolve readily once the temperature is lowered again. Thermoresponsive bioconjugates carrying an affinity ligand have been suggested for the specific purification of biologicals by affinity precipitation. The involved bioconjugate is called an affinity macroligand, AML. The polymer mediates the response to the stimulus (e.g. a change in temperature). The affinity tag enables the AML to attach itself to the target molecules. Affinity precipitation is to date not an established downstream processing technique in the biotech industry. The objective of this thesis was to find potential applications for the affinity precipitation by first choosing interesting target molecules and second by comparing the affinity precipitations with well established affinity bioseparation procedures based on chromatography or magnetic beads. The biomolecules chosen as target products were poly(A) mRNA, scFv antibody phages, hemoglobin and IgG 4E