Knowledge-driven strategies for process development in protein refolding

Abstract

Escherichia coli has been a relevant host organism for the production of heterologous proteins. Several advantages like cheap cultivation media, fast replication rates and its well-studied genome made E. coli especially interesting for industrial applications. However, overexpression of the target protein leads to its accumulation in insoluble aggregates called inclusion bodies (IBs). For a long time IBs were considered waste products as they are known to contain inactive and misfolded protein. As it was found that the bioactive protein can be recovered by downstream processing, nowadays, protein production in IBs is desired as it bares the possibility of achieving high yield processes at an increased protein purity within a short time-frame. The extensive downstream process for refolding of IBs consists of various process units including isolation, solubilization, refolding and concentration of the product. The commonly applied downstream process, a batch or fed-batch dilution approach, was found to result in low product yields and productivities leading to a reduced efficiency of the entire production process. The refolding unit operation is especially prone to high losses due to several limitations like reaction kinetics that lead to aggregate formation, a lack of suitable analytics at low product concentrations or adequate mixing at high volumes. As online monitoring tools and process control strategies are lacking, a reproducible and controlled refolding environment cannot be provided. Additionally, as there is no versatile platform technology for protein recovery from IBs, the entire processing conditions need to be reevaluated for every new application leading to immense efforts in process development.The hypothesis to solve this problem is that a time revolved description of the refolding kinetics along with a digital twin, which captures the understanding of the kinetics as well as the interaction between the process parameters and the quality attributes, will be the enabler to provide a platform technology for refolding. Thus, evaluation and experimental analysis of the individual process parameters during protein refolding and their interdependencies should give more insights into their influence on efficient downstream processing. Finding suitable analytical tools to observe the ongoing kinetics and to quantify low product quantities is essential to understand the dynamics of protein refolding. Process analytical technology (PAT) should be used in combination with control strategies based on process models to establish robust refolding processes with elevated product yield and productivity.