Novel hypoxia responding cell system for substantial improvement of recombinant protein production

Novel hypoxia responding cell system to overcome the limitation of oxygen supply in high-density cell cultures for improved recombinant protein production.

The increase of biomolecule production under hypoxic conditions is particularly advantageous for cell cultures with high cell densities, in which low levels of oxygen are inherently present.

• Increased product yield: up to ten-fold induction of the protein production
• Enhancing the robustness of production process as decreasing oxygen level lead to improved  productivity  
• Reduced costs of biomolecule production as gassing rate can be reduced or even eliminated at the end of the process
• Allows reduction of the stirring speed, which significantly reduces shear stress acting on the cells
• Improving the ecological sustainability (better environmental balance at lower costs)
• Equal or improved product quality

Batch and intensified fed-batch fermentation as well as high-density perfusion processes to produce biomolecules like therapeutic proteins and antibodies.

A further attractive option enabled by the utilization of HREs is the application of novel bioprocessing strategies for discontinuous fed-batch processes. Using a HREs containing vector system, hypoxia could be exploited by adjustment of oxygen setpoints after cells reach the static phase to induce and drive hypoxia responsive expression of the recombinant protein.

The biotechnological production of biopharmaceuticals using cell cultures is playing an increasingly important role, especially in the field of therapeutic proteins and antibodies. The production of these proteins is usually carried out using eukaryotic expression cell lines, including in particular "chinese hamster ovary cells" (CHO cells) as by far one of the most important mammalian cell lines. The cells are industrially grown in suspension in large numbers to obtain high yields of the biopharmaceutical active ingredient. However, this process is expensive, laborious and time-consuming due to various factors such as properties and stability of the product as well as various process challenges. Numerous methods and techniques have been developed over time to increase production and protein yield. These include technical improvements in plant control (such as control and adjustment of pH, O₂ levels, feeding of nutrients, etc.), but also optimization of the cells or cell lines themselves to improve their survivability and production performance. Genetic modifications in particular have led to a strong increase in specific and volumetric productivity, e.g. through optimization of cell lines or targeted activation of protein production.

During cellular production of biotherapeutic proteins in classical batch fermentations and perfusion processes, sufficient oxygen supply is essential for cellular respiration and cellular functions. However, at high cell densities or with large volumes, the supply of oxygen to the cells is difficult, resulting in hypoxic conditions that reduce cell proliferation, viability and productivity. Since industrially used CHO cells are not optimized for protein production under hypoxia, cell engineering to further improve this dominating production system is needed.

A novel hypoxia responding cell system based on hypoxia-responsive elements (HRE) and cellular oxygen sensing proteins was developed for CHO cells. HRE are regulatory enhancer elements that have been identified in the promoter region of numerous oxygen-responsive genes such as VEGF-A, EPO and other genes. The HREs serve as the binding site for hypoxia-inducible factors, which are transcription factors that mediate the adaptation of cells to low oxygen levels (hypoxia) by enhancing the transcription of the respective genes. It was found that the HRE sequence of VEGF-A gene can be exploited for increasing the production of a biomolecule under hypoxic conditions. To do so, at least one HRE of the VEGF-A gene is operably combined with a suitable promoter and the nucleic acid sequence encoding the biomolecule of interest. This leads to an increased transcription of the nucleic acid sequence encoding the biomolecule under conditions of oxygen deficiency and thus to an increased production of the biomolecule. In addition, a correlation between HRE number and expression response was observed, which was saturated between 5 and 8 HRE repetitions. The amount of biomolecule produced by the hypoxia-inducible cells was nearly 10-fold higher than the amount produced by non-hypoxia-inducible control cells under hypoxic conditions.

Publication of WO2022243320A1

Zeh et al., Exploiting hypoxia in CHO for improved productivity during high cell density cultivation and novel bioprocessing strategies, Metabolic Engineering Communications, Vol 13, 2021.

Zeh et al., Exploring synthetic biology for the development of a sensor cell line for automatedbioprocess control. Sci Rep. 2022 Feb 10;12(1):2268.

https://www.hochschule-biberach.de/research-group-kerstin-otte