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.

A roadmap for the synthesis of separation networks for the recovery of bio-based chemicals: Matching biological and process feasibility

“…The suitability grades for pretreatment and cell harvesting technologies are the same while there are some variations for cell disruption and phase isolation options. HPH (high pressure homogenization) could be a good choice as it has been used previously for IN-SOL products (Balasundaram et al, 2011). Soluble products are more susceptible to chemical interactions by acids and alkalis, hence chemical lysis may not be suitable in many cases.…”

High throughput screening of particle conditioning operations: I. System design and method development

“…Solubility screening with precipitation was evaluated by several groups (Ahmad and Dalby, ; Kramarczyk et al, ; Nfor et al, ; Wiendahl et al, ; Yoshimoto et al, ). More recently, reports have aimed at optimizing precipitation and flocculation conditions for the recovery of mAbs and Fc‐fusion proteins (Balasundaram et al, ; Knevelman et al, ; Ma et al, ; Sheth et al, ). These studies employed a broad range of precipitation agents and conditions to identify regions of superior yield, quality, and impurity clearance.…”

High throughput screening of particle conditioning operations: II. Evaluation of scale‐up heuristics with prokaryotically expressed polysaccharide vaccines

“…The advent of high throughput process development (HTPD) has created opportunities for streamlining purification development of biological candidates (Titchener‐Hooker et al, ). Much progress has been made in designing high throughput methods for the purification optimization of a single protein with microfiltration, disruption, extraction, solubilization, refolding, centrifugation, precipitation, filtration, and chromatography (Aucamp et al, ; Balasundaram et al, ; Chhatre and Titchener‐Hooker, ; Coffman et al, ; Kong et al, ; Peng et al, ; Tait et al, ; Treier et al, ; Tustian et al, ; Walther et al, ; Wenger et al, , ). Many authors have shown how data attained with HTPD can be used to predict performance at much larger scales.…”