Process engineering of biopharmaceutical production in moss bioreactors via model-based description and evaluation of phytohormone impact

The moss Physcomitrella is an interesting production host for recombinant biopharmaceuticals. Here we produced MFHR1, a synthetic complement regulator which has been proposed for the treatment of diseases associated to the complement system as part of human innate immunity. We studied the impact of different operation modes for the production process in 5 L stirred-tank photobioreactors. The total amount of recombinant protein was doubled by using fed-batch or batch compared to semi-continuous operation, although the maximum specific productivity (mg MFHR1/g FW) increased just by 35%. We proposed an unstructured kinetic model which fits accurately with the experimental data in batch and semi-continuous operation under autotrophic conditions with 2% CO2 enrichment. The model is able to predict recombinant protein production, nitrate uptake and biomass growth, which is useful for process control and optimization. We investigated strategies to further increase MFHR1 production. While mixotrophic and heterotrophic conditions decreased the MFHR1-specific productivity compared to autotrophic conditions, addition of the phytohormone auxin (NAA, 10 μM) to the medium enhanced it by 470% in shaken flasks and up to 230% and 260%, in batch and fed-batch bioreactors, respectively. Supporting this finding, the auxin-synthesis inhibitor L-Kynurenine (100 μM) decreased MFHR1 production significantly by 110% and 580% at day 7 and 18, respectively. Expression analysis revealed that the MFHR1 transgene, driven by the Physcomitrella actin5 (PpAct5) promoter, was upregulated 16 hours after NAA addition and remained enhanced over the whole process, whereas the auxin-responsive gene PpIAA1A was upregulated within the first two hours, indicating that the effect of auxin on PpAct5 promoter-driven expression is indirect. Furthermore, the day of NAA supplementation was crucial, leading to an up to 8-fold increase of MFHR1-specific productivity (0.82 mg MFHR1/ g fresh weight, 150 mg accumulated over 7 days) compared to the productivity reported previously. Our findings are likely to be applicable to other plant-based expression systems to increase biopharmaceutical production and yields.

Biopharmaceutical-Type Chinese Hamster Ovary Cell Cultivation Under Static Magnetic Field Exposure: A Study of Genotoxic Effect

The lack of a sufficient research base is the reason for the ongoing discussion regarding the genotoxic effect of magnetic field (MF) exposure on mammalian cell cultures. Chinese hamster ovary (CHO) suspension-type cells, which are widely used for biopharmaceutical production, are potentially subjected to an increased MF when cultivated in bioreactors equipped with bottom-placed magnetically coupled stirring mechanisms. The main challenge for conducting research in this field remains the availability of a suitable experimental setup that generates an appropriate MF for the raised research question. In the present study, a simple and cost-effective experimental setup was developed that generated a static MF, similar to what has been modeled in large-scale bioreactors and, at the same time, was suitable for experimental cell cultivation in laboratory conditions. The measured maximum magnetic flux density to which the cells were exposed was 0.66 T. To assess the possible genotoxic effect, cells were continuously subcultivated in laboratory petri dishes for a period of 14 days, corresponding to a typical duration of a biopharmaceutical production process in a conventional fed-batch regime. The genotoxic effect was assessed using the cytokinesis-block micronucleus assay with fluorescent staining. Results showed that a 0.66-T static MF exposure had no significant long-term effect on cell viability and chromosomal damage but demonstrated a short-term effect on cell apoptosis. Significant increase in nuclear bud formation was observed. These findings may encourage other researchers in future studies investigating cellular responses to MF exposure and contribute relevant data for comparison.

Strategies for Enhancing Product Yield: Design of Experiments (DOE) for Escherichia coli Cultivation

E. coli is considered one of the best model organism for biopharmaceutical production by fermentation. Its utility in process development is employed to develop various vaccines, metabolites, biofuels, antibiotics and synthetic molecules in large amounts based on the amount of yield in shake flasks, bioreactors utilised by batch, fed-batch and continuous mode. Production of the desired molecule is facilitated in the bioreactor by employing strategies to increase biomass and optimised yield. The fermentation is a controlled process utilising media buffers, micronutrients and macronutrients, which is not available in a shake flask. To maximise the production temperature, dissolved oxygen (aerobic), dissolved nitrogen (anaerobic), inducer concentration, feed or supplementation of nutrients is the key to achieving exponential growth rate and biomass. Design of experiments (DOE) is critical for attaining maximum gain, in cost-effective manner. DOE comprises of several strategies likewise Plakett-Burman., Box–Behnken, Artificial Neural Network, combination of these strategies leads to reduction of cost of production by 2–8 times depending on molecules to be produced. Further minimising downstream process for quickly isolation, purification and enrichment of the final product.

Strategies for the Production of Soluble Interferon-Alpha Consensus and Potential Application in Arboviruses and SARS-CoV-2

Biopharmaceutical production is currently a multibillion-dollar industry with high growth perspectives. The research and development of biologically sourced pharmaceuticals are extremely important and a reality in our current healthcare system. Interferon alpha consensus (cIFN) is a non-natural synthetic antiviral molecule that comprises all the most prevalent amino acids of IFN-α into one consensus protein sequence. For clinical use, cIFN is produced in E. coli in the form of inclusion bodies. Here, we describe the use of two solubility tags (Fh8 and DsbC) to improve soluble cIFN production. Furthermore, we analyzed cIFN production in different culture media and temperatures in order to improve biopharmaceutical production. Our results demonstrate that Fh8-cIFN yield was improved when bacteria were cultivated in autoinduction culture medium at 30 °C. After hydrolysis, the recovery of soluble untagged cIFN was 58% from purified Fh8-cIFN molecule, fourfold higher when compared to cIFN recovered from the DsbC-cIFN, which achieved 14% recovery. The biological activity of cIFN was tested on in vitro model of antiviral effect against Zika, Mayaro, Chikungunya and SARS-CoV-2 virus infection in susceptible VERO cells. We show, for the first time, that cIFN has a potent activity against these viruses, being very low amounts of the molecule sufficient to inhibit virus multiplication. Thus, this molecule could be used in a clinical approach to treat Arboviruses and SARS-CoV-2.