title: Dual salt precipitation for the recovery of a recombinant protein from Escherichia coli.
abstract: When considering worldwide demand for biopharmaceuticals, it becomes necessary to consider alternative process strategies to improve the economics of manufacturing such molecules. To address this issue, the current study investigates precipitation to selectively isolate the product or remove contaminants and thus assist the initial purification of a intracellular protein. The hypothesis tested was that the combination of two or more precipitating agents will alter the solubility profile of the product through synergistic or antagonistic effects. This principle was investigated through several combinations of ammonium sulfate and sodium citrate at different ratios. A synergistic effect mediated by a known electrostatic interaction of citrate ions with Fab' in addition to the typical salting-out effects was observed. On the basis of the results of the solubility studies, a two step primary recovery route was investigated. In the first step termed conditioning, post-homogenization and before clarification, addition of 0.8 M ammonium sulfate extracted 30% additional product. Clarification performance measured using a scale-down disc stack centrifugation mimic determined a four-fold reduction in centrifuge size requirements. Dual salt precipitation in the second step resulted in >98% recovery of Fab' while removing 36% of the contaminant proteins simultaneously.
authors.affiliation: Dept. of Biochemical Engineering, The Advanced Centre for Biochemical Engineering, University College London, London WC1E 7JE, UK.
authors.forename: Bangaru
authors.initials: B
authors.lastname: Balasundaram
authors.forename: Soam
authors.initials: S
authors.lastname: Sachdeva
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Bracewell
mesh_terms: D011232:Chemical Precipitation; D004926:Escherichia coli / Q000235:genetics*; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000302:isolation & purification; D010316:Particle Size; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012492:Salts; D012995:Solubility; D055672:Static Electricity
chemical_list: D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D012492:Salts