Primary Recovery of Biologically Active Compounds Using Macroporous Monolithic Hydrogels

Sammanfattning: The completion of sequencing of human and other mammalian genomes opens a new era for drug development. New approaches have been proposed for tackling diseases such as development of small molecule therapies based on understanding of human genome and proteome, new proteinous based therapies, where the therapeutic protein can inhibit or modify a certain metabolic pathway or DNA-based therapies delivering a gene into the cell (e.g., cancer cells) to correct or inhibit the imperfect gene function. The possibility of production of novel therapeutics (small, proteinous and DNA based substances) in biotechnological processes utilising mammalian cells or microorganisms such as bacteria or yeast, presents new challenges for the separation processes. Moreover, new methods for high throughput screening are needed to identify potentially important lead molecules within the large molecular libraries. Indeed, designing downstream process (DSP) of biologically active compounds depends on the nature and intended use of such compounds. Commodity compounds that are produced in tons need fast and cheap purification methods. However, high molecular weight molecules intended for pharmaceutical applications, need careful design of DSP to meet the legislator demands such as removal of contaminants and toxic substances. Two examples were demonstrated for the design of DSP purification process for small and large molecular weight compounds. Lactic acid was purified from cell broth using composite ion exchanger in expanded bed chromatographic format. Plasmid DNA was captured directly from cell lysate using monolithic macroporous hydrogel, so called cryogel, grafted with different polycations. Using cryogels with coupled specific ligands, bacterial endotoxin was successfully removed from protein solutions and captured from disposed cell homogenate. Fast parallel screening of complex samples containing target substance was demonstrated using affinity minicryogel columns in 96-well format plate.