The economics of inclusion body processing

Abstract

Many recombinant proteins are often over-expressed in host cells, such as Escherichia coli, and are found as insoluble and inactive protein aggregates known as inclusion bodies (IBs). Recently, a novel process for IB extraction and solubilisation, based on chemical extraction, has been reported. While this method has the potential to radically intensify traditional IB processing, the process economics of the new technique have yet to be reported. This study focuses on the evaluation of process economics for several IB processing schemes based on chemical extraction and/or traditional techniques. Simulations and economic analysis were conducted at various processing conditions using granulocyte macrophage-colony stimulating factor, expressed as IBs in E. coli, as a model protein. In most cases, IB processing schemes based on chemical extraction having a shorter downstream cascade demonstrated a competitive economic edge over the conventional route, validating the new process as an economically more viable alternative for IB processing.

Production of recombinant proteins in E. coli by the heat inducible expression system based on the phage lambda pL and/or pR promoters

“…gr., protein sequence), growth and induction conditions, cytoplasmic reducing environment, kinetic competition between aggregation and folding, high local protein concentration, inappropriate interactions with in vivo folding chaperones, intermolecular disulfide crosslinking (although proteins lacking cysteines can still form inclusion bodies), medium composition, and bacterial strain [137,150-153]. Accumulation of the recombinant protein in IB has certain advantages, as it can be isolated and concentrated by a simple centrifugation step, reducing the downstream processing costs and facilitating the production of toxic proteins to cells [154-156]. However, a refolding step is then required to recover a biologically active protein, and refolding steps can be inefficient [157,158].…”

Recent advances in biomolecular process intensification

“…It is widely accepted that refolding cost dominates overall processing cost when the recombinant proteins are expressed as IBs in E. coli. While this is true in traditional IB processing schemes in which the refolding step is dominated by dilution/dialysis of denatured-solubilised proteins, process economics analysis showed that the cost associated with pre-refolding section accounts for a significant proportion of overall production cost when refolding is conducted in a matrix-assisted on-column refolding reactor with a moderate concentration of denatured protein (Lee et al, 2005). This illustrates the need to redesign the pre-refolding section of the IB processing scheme and to consider the IB processing scheme in its entirety to achieve process intensification.…”

“…Although spermine proved very effective in the selective removal of DNA, the relatively high cost of spermine may not justify its use at large scale. However, the use of costeffective DNA precipitants such as polyethyleneimine (PEI) is well documented (Burgess, 1991;Helander et al, 1997), and likely to be effective in replacing spermine without compromising the selectivity or efficiency for DNA removal (Lee et al, 2005).…”

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

“…6,10,34 Batch dilution refolding is the most widely used method for protein refolding, and it is currently used at an industrial scale. Typically, batch dilution refolding operates at relatively low total protein concentration (0.01-0.10 mg/mL), 8,9 to slow the protein aggregation rates down, favoring the refolding yield. Accordingly, a significantly high dilution is needed resulting in large processing volumes, requiring large vessels and subsequent concentration steps.…”

“…To attain relatively high refolding yields, generally the total protein concentration after dilution is set to a value in the range of 0.01-1 mg/mL. [8][9][10][11] This relatively low protein concentration primarily slows down the rates of protein aggregation, which is competing against the first-order refolding reaction, positively influencing the refolding yield. However, operating at such low protein concentration implies significant dilutions, resulting in large processing volumes, large vessels, and extensive product concentration steps.…”