Bilfinger Industrietechnik Salzburg

Navigation
Meldung

Controlled refolding of inclusion body protein to boost product yields

28.02.2018

In cooperation with TU Wien (University of Technology Vienna), Bilfinger Industrietechnik Salzburg designed a refolding vessel in pilot scale capable of controlling process parameters, which are crucial for a successful refolding process. Applying controlled conditions during refolding leads to a robust, reproducible and scalable process as recommended by the ICH Harmonised Tripartite Guidelines for Pharmaceutical Development Q8. In addition, higher product yields can be obtained and by applying Fed-Batch refolding the space time yields is increased. This novel device presents a platform tool for refolding of a huge variety of different products.

Escherichia coli is the most widely used host for the production of biopharmaceuticals as it can grow on cheap substrates and yields high biomass and product titers. One disadvantage of this production host is the formation of so called inclusion bodies (IBs). IBs are agglomerates of misfolded protein which have a high purity, but mostly no activity. Therefore, those IBs need to be solubilized using denaturing reagents and subsequently refolded to obtain product in its native conformation.

After solublization of the protein, an uncontrolled dilution approach is carried out for refolding, which requires big vessels and leads to low product titers due to agglomeration of protein and formation of wrongly folded species.

Bilfinger´s solution for more efficient refolding processes

To create an optimum environment for the protein to refold and yield high product titers, we monitor and control several parameters during the refolding process. Amongst those the most important parameters are the following

  • Temperature: Folding and aggregation of proteins can be impacted by the temperature
  • pH: Disulfide bond formation is usually favored at basic pH
  • Dissolved Oxygen and Redox Potential: An adequate balance between reductive and oxidant species is necessary to optimize the oxidative folding as disulfide bonds undergo reduction followed by oxidative rearrangement to form the native protein structure

Furthermore we boost productivity by Fed-batch Refolding to increase the space time yield. Solubilized protein is continuously added to the refolding vessel by a controlled feeding strategy. Thus, refolding can be performed at higher protein concentration and a reduced vessel size.