Affiliation: | a Laboratório de Engenharia de Bioprocessos, Departamento de Processos Biotecnológicos, Faculdade de Engenharia Química, Universidade Estadual de Campinas, CP 6066, CEP 13083-970 Campinas, SP, Brazil b Laboratório de Interação Molecular e Bioengenharia, Departamento de Processos Biotecnológicos, Faculdade de Engenharia Química, Universidade Estadual de Campinas, CP 6066, CEP 13083-970 Campinas, SP, Brazil |
Abstract: | Aprotinin, a bovine protease inhibitor currently also produced in recombinant bacteria, yeast, and corn, has valuable applications as a human therapeutic and in tissue culture. The objective of this work was to develop the basis of a large-scale aprotinin purification process centered on immobilized metal ion affinity chromatography (IMAC). This technique uses ligands—metal ions—of a lower cost and higher stability than those traditionally used in affinity chromatography. Since aprotinin does not interact with IMAC ligands, collection is from the nonretained fractions (negative chromatography). Stirred-tank batch IMAC adsorption experiments indicated that one-step aprotinin purification could not be successful. Immobilized chymotrypsin chromatography was then used as a prepurification step, yielding a suitable feed for IMAC (with purification factors as high as 476). IMAC column fed with these prepurified materials produced purified aprotinin in the nonretained fractions with purification factors as high as 952. |