A rapid high recovery diafiltration process for the separation and purification of biologically active proteins and peptides will be developed and demonstrated. The mild process conditions and size selectivity of membrane filtration will be used to recover biologically produced fusion proteins of 15,000 to 20,000 molecular weight. The fusion protein will be constructed to contain the desired biologically active peptide or protein and a protein of greater than 10,000 daltons that in a fusion gene construct negates the bioactive to the host systems. A new high flux ultrafiltration hollow fiber will be employed in two early diafiltration steps to first remove all large species; greater than 25 to 30,000 daltons; (i.e. cells, cell debris, enzymes, etc.) and second to recover and concentrate the fusion protein while eliminating small species of less than 10,000 daltons composed of metabolites, nutrient medium, etc. This small volume of over 100 fold fusion protein concentrate may then selectively be cleaved and further processed employing diafiltration to obtain a pure bioactive product with good yields. The process will be demonstrated using a biological active natural peptide; cecorpin or an analog.
