CD Laboratory for Inclusion Body Processing 4.0

This CD Laboratory is researching the fundamentals for the targeted production of so-called inclusion bodies, product aggregates that are produced during the production of recombinant proteins in E. coli and have great potential as biopharmaceuticals.

E. coli is an established production organism for recombinant proteins in the biopharmaceutical industry. During this process, insoluble product aggregates, so-called inclusion bodies (IBs), are also formed. It has been shown that these IBs have extremely attractive properties and can be utilised for the production of biopharmaceuticals. For example, IBs have extremely high concentrations of active ingredients (product titre) and very high product purity. Nevertheless, state-of-the-art processes for the targeted production of IBs are still based on empiricism rather than quality by design (QbD). Processes for the controlled dissolution (solubilisation) and refolding of proteins from IBs are developed specifically for individual products. There is no platform knowledge from which general principles can be derived. In addition, these processes are characterised by low yields, high variability, unpredictability, long development times and environmental problems.

In this CD Laboratory, the basic aspects of how to obtain protein secondary structures in IBs are investigated, i.e. how hydrogen bonds between the CO and NH groups of the peptide backbone can be used to achieve targeted formation, for example of a helix. The protein-specific limitations in this respect are also determined. Furthermore, the expression systems will be compared and analysed to determine how E. coli reacts to various process parameters with regard to physiology, viability, productivity and product quality.

In addition, possibilities for efficient IB harvesting are being investigated, i.e. the conditions under which active protein can be released from the insoluble IBs are being clarified. For this purpose, standing ultrasound waves and ionic liquids are available as a novel mild solubilisation agent. Furthermore, the effect of electrical pulses on the refolding of the released protein will be investigated and existing gaps in the basic knowledge of refolding models will be closed.

The short-term goals of this CD Laboratory are to expand the basic understanding of IB formation and to develop novel methods and tools for processing and monitoring. The process understanding gained will help to reduce the environmental footprint of such processes by reducing buffer volumes and reactor sizes. The medium-term goal is to convert the current manufacturing process based on individual batches into a (semi-)continuous process with corresponding process analytics, thus enabling smaller plants. The long-term goal is to analyse the entire IB process from cultivation in the bioreactor through to protein refolding and product purification, thereby generating detailed knowledge about each process step. Process understanding is created and described by means of solid data evaluation and modelling in order to ultimately obtain a digital twin of selected IB processes and thus make them fit for "Industry 4.0". The basic knowledge gained about IBs can ultimately be transferred to industry.