Refolding of biotech therapeutic proteins expressed in bacteria: review

Abstract

The efficiency of the protein refolding process lies in identification of the optimal conditions. However, a number of challenges need to be overcome to achieve this. This review first describes the protein refolding process that is utilized presently for production of protein therapeutics. Next, it discusses the various shortcomings that exist with respect to the present approach. The focus of the paper is on presentation of the significant advancements that have been made in the past decade in the various aspects of protein folding, including use of bioinformatics, mechanistic modeling, analytical monitoring, process optimization, use of additives, high throughput development, on‐column refolding, Quality by Design (QbD), Process Analytical Technology (PAT), and process intensification. Finally, an approach is proposed that incorporates the best practices that have been identified in the various areas. The paper is expected to be of interest to those in academia and industry working in the area of protein refolding. © 2013 Society of Chemical Industry.

Solubilization of inclusion body proteins using low and very low concentrations of chemicals: implications of novel combined chemical treatment designs in enhancement of post‐solubilization target protein purity and biological activity

“…Accordingly, such a solubilization method can make the downstream processing of the protein much shorter and cheaper. Refolding of rSK subsequent to IBs solubilization has been done by various methods such as dilution and dialysis, none of which looks appropriate for large‐scale application compared with gel filtration used for refolding in the present work.…”

Efficient refolding of the bifunctional therapeutic fusion protein VAS-TRAIL by a triple agent solution

“…coli remains the most popular and cost-effective host for producing recombinant proteins not only for biochemical research but also for pharmaceutical applications [25]. However, recombinant protein expression in E. coli often leads to the formation of insoluble aggregates known as IBs [26]. As a result, the production of therapeutic proteins in E. coli sometimes requires the conversion of the aggregated protein into the correctly folded, three-dimensional structure with the required therapeutic activity.…”

Wanted: more monitoring and control during inclusion body processing

“…Finally, the refolded POI might undergo a purification step to further increase purity. A more detailed IB process description can be found elsewhere (Basu et al 2011; Burgess 2009; Eggenreich et al 2016; Hoffmann et al 2017; Kaur et al 2017; Rathore et al 2013; Singh et al 2015). …”

“…A major problem are non-competitive yields because of low recovery rates. Commercial IB processes often employ an uncontrolled dilution method with immense buffer volumes requiring huge refolding vessels (Rathore et al 2013). It is known that mixing times increase with scale because uniform stirring is challenging in big tanks.…”