Techno‐economic evaluation of an inclusion body solubilization and recombinant protein refolding process

Abstract

Expression of recombinant proteins in Escherichia coli is normally accompanied by the formation of inclusion bodies (IBs). To obtain the protein product in an active (native) soluble form, the IBs must be first solubilized, and thereafter, the soluble, often denatured and reduced protein must be refolded. Several technically feasible alternatives to conduct IBs solubilization and on-column refolding have been proposed in recent years. However, rarely these on-column refolding alternatives have been evaluated from an economical point of view, questioning the feasibility of their implementation at a preparative scale. The presented study assesses the economic performance of four distinct process alternatives that include pH induced IBs solubilization and protein refolding (pH_IndSR); IBs solubilization using urea, dithiothreitol (DTT), and alkaline pH followed by batch size-exclusion protein refolding; inclusion bodies (IBs) solubilization using urea, DTT, and alkaline pH followed by simulated moving bed (SMB) size-exclusion protein refolding, and IBs solubilization using urea, DTT and alkaline pH followed by batch dilution protein refolding. The economic performance was judged on the basis of the direct fixed capital, and the production cost per unit of product (P(C)). This work shows that (1) pH_IndSR system is a relatively economical process, because of the low IBs solubilization cost; (2) substituting β-mercaptoethanol for dithiothreithol is an attractive alternative, as it significantly decreases the product cost contribution from the IBs solubilization; and (3) protein refolding by size-exclusion chromatography becomes economically attractive by changing the mode of operation of the chromatographic reactor from batch to continuous using SMB technology.

Application of process analytical technology for downstream purification of biotherapeutics

“…These inclusion bodies need to be solubilized using chaotropic/reducing agents to disrupt noncovalent interactions and break the interchain disulfide bonds. Subsequently, in the refolding step, the concentration of the solubilizing agents is decreased, resulting in protein refolding . This can be achieved by buffer exchange via batch dilution or liquid chromatography.…”

Chaotropic heat treatment resolves native‐like aggregation of a heterologously produced hyperthermostable laminarinase

“…). Refolding is often achieved by diluting the denaturant through addition (or exchange) of buffer, or by using liquid chromatography (chromatographic refolding) like size‐exclusion chromatography . High pressure treatment, either in the absence or presence of chaotropic agent, can also be useful to foster refolding of proteins from inclusion bodies .…”

“…Using a chaotropic agent, like urea or guanidine hydrochloride (GuHCl), a reducing agent to break disulfide bonds, and alkaline pH, commonly solubilizes inclusion bodies (see e.g. ). Refolding is often achieved by diluting the denaturant through addition (or exchange) of buffer, or by using liquid chromatography (chromatographic refolding) like size‐exclusion chromatography .…”

Oxidative protein refolding on size exclusion chromatography at high loading concentrations: Fundamental studies and mathematical modeling

“…For these reasons SEC has been widely used at lab scale for protein refolding in either batch or continuous mode using single or multiple column configurations (i.e. simulated moving bed) [1][2][3][4][5][6][7][8][9]. Existing work has also developed mathematical models for separation in SEC using various model proteins in their native forms [10,11].…”

“…Conventional method of protein refolding, namely batch dilution refolding, requires working at low protein concentrations in order to prevent aggregation, which in turn results in low productivity impeding high-throughput protein refolding and downstream processing for production of many bacterially expressed recombinant proteins [1]. Size exclusion chromatography (SEC)-based protein refolding addresses this issue to some extent by facilitating gradual spatial isolation of protein molecules and unfolding agents, which can prevent aggregation and allow for application of higher protein loading concentrations and simultaneous purification compared with batch dilution refolding.…”