Inclusion Body Processing 4.0

CD Laboratory for Inclusion Body Processing 4.0

[Translate to English:] Logos der TU Wien - IBD Group und Boehringer Ingelheim

Laboratory Head: Assoc Prof. Oliver Spadiut

Academic Partner: TU Wien

Industrial Partner: Boehringer Ingelheim RCV GmbH & Co KG


Escherichia coli is one of the most widely used hosts to produce recombinant proteins. Its well-known genetics, the great variety of expression systems and production strains and the low cultivation costs as well as short generation times and high product titers make it very attractive for basic research as well as manufacturing purposes (e.g., [1-5]). To date, more than 25% of all biopharmaceuticals are produced in E. coli [3, 6-8]. However, a common consequence of overproduction of recombinant protein in E. coli is the formation of insoluble product aggregates, called Inclusion Bodies (IBs) (Figure 1; e.g., [4, 7, 9-12]). Inclusion Bodies occur in the polar region of the bacterium and are characterized by a porous structure, spherical or rod-shaped appearance and a diameter of up to around 700-800 nm [11, 13-19].

[Translate to English:] Inclusion Bodies Pictures from TEM and SEM

Figure 1: Inclusion Bodies (IBs). A, TEM picture showing IBs in recombinant E. coli cells; B, SEM picture of isolated IBs on a filter. Both pictures were taken at TU Wien in 2020.

Until around 20 years ago, IBs were considered as non-functional waste products and various strategies were developed to reduce or completely avoid their formation (e.g., [11, 20-28]). However, approximately 80% of all recombinant proteins overexpressed in E. coli still form IBs [29]. In the last 20 years, numerous studies have demonstrated that proteins enclosed in IBs can be biologically active making IB formation an attractive strategy to produce large amounts of recombinant protein (e.g., [18, 30-48]). Thus, several direct applications of bioactive IBs in biocatalysis, synthetic chemistry and biomedicine have been developed [16, 38-42, 49, 50]. These bioactive IBs are used as immobilized biocatalysts [40, 41, 51-53], scaffolds and functional materials in tissue engineering [54-59], targeted and non-targeted drug delivery systems [57, 60, 61] as well as depots of therapeutic proteins, called nanopills [62, 63]. The importance of bioactive protein aggregates has recently been underlined by the development of pull-down tags, which induce IB formation of otherwise soluble proteins via intermolecular aggregation (e.g., [40, 64-73]).

Besides the direct use of catalytically active IBs, the fast-emerging market of biosimilars in the biopharmaceutical industry has a high demand for recombinant therapeutic proteins. Therefore, to ensure steady medical supply and enable economic manufacturing, it is of the utmost importance to enhance yield, accelerate process development time and lower production costs. Considering these aspects, the production of recombinant biopharmaceuticals in form of IBs instead of soluble protein presents a highly attractive option with many advantages  [7, 14, 19, 45, 46, 74-82], such as high product yield (more than 10 g IBs/L cultivation broth), high product purity in IBs (up to 95%), high mechanical and thermal stability of IBs, resistance of IBs to proteases, easy isolation of IBs due to differences in size and density compared to host cell proteins, IBs contain native and native-like secondary protein structures and presence of biological activity.


Still, the current challenges for state-of-the-art IB processes are:

  • To create a universal platform strategy rather than case-by-case approaches
  • To miniaturize and automate process development
  • To establish alternatives to harsh solubilization (e.g., extraction of soluble product from IBs, mild solubilization [143] or combinatorial strategies)
  • To reduce buffer/water consumption and vessel size in refolding processes and thus reduce the environmental footprint
  • To generate process knowledge and understanding
  • To digitalize and pave the way to Industry 4.0

Within this CD Laboratory entitled “Inclusion Body Processing 4.0” we aim to address the listed challenges of state of the-art IB based processes, trying to pave the way to an universal, economic platform process, using sophisticated analytical and processing methods.

[Translate to English:] Overview of the CD Laboratory „Inclusion Body Processing 4.0“.

Figure 2: Overview of the CD Laboratory „Inclusion Body Processing 4.0“.


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