Process Engineering of Biopharmaceutical Production in Moss Bioreactors via Model-Based Description and Evaluation of Phytohormone Impact.

Ralf Reski,Sina Schroeder,Clemens Posten,Juliana Parsons,Eva L Decker,Natalia Ruiz-Molina

doi:10.3389/fbioe.2022.837965

Abstract

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 h after NAA addition and remained enhanced over the whole process, whereas the auxin-responsive gene PpIAA1A was upregulated within the first 2 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.

Highlights

There are technical advantages associated with up-stream processing: For instance, a rapid scale-up of production using Nicotiana benthamiana and a transient expression system is feasible within 2 months (Shoji et al, 2012; Capell et al, 2020), which would allow a quick response to address a public health crisis
We explored the effect of process operating mode on MFHR1 production as a means for targeted changes of environmental conditions for the cells
Based on these experiments we propose a kinetic model of the moss bioreactor which can accurately predict recombinant protein production, biomass growth and nitrate uptake under batch and semi-continuous operation, and can be used to maximize protein and biomass productivity

Summary

Introduction

The production of biopharmaceuticals, which include enzymes, vaccines, antibodies, growth factors, and hormones, is a complex task where small differences in production conditions can influence product quality, efficacy, safety and yield. Plant-based biopharmaceuticals have gained increasing attention as an alternative to mammalian cell systems in the last decade; only β-. One advantage of plant-based expression systems is biosafety due to lack of contamination with animal-borne viruses. During the current COVID-19 pandemic, it became evident that diagnostic reagents and vaccine production capacity was not sufficient to meet demand, and transient expression in plants could compensate this (Tusé et al, 2020). The plant-derived virus-like particles vaccine candidate for COVID-19 by Medicago completed phase I clinical trials with promising results and the phase II/III trials are ongoing (Gobeil et al, 2021; Ward et al, 2021). There are many plant-based vaccines in phase I clinical trials (Shim et al, 2019), and some of them completed phases II and III such as avian (monovalent) and seasonal (quadrivalent) influenza vaccines produced in N. benthamiana (Ward et al, 2020)

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