A synthetic protein as efficient multitarget regulator against complement over-activation

Front. Bioeng. Biotechnol.

Schroeder

et al. 2022

Self Cite

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.

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Ruiz-Molina

Front. Bioeng. Biotechnol.

Schroeder

et al. 2022

Self Cite

“…The single copy gene PpCLF (Pp3c22_22940V3.1) was used as a reference to normalize the data using two primer pairs listed in Supplementary Table S1. The number of transgene copies was determined using 2 controls as calibrators, a moss line with a single integration of the hygromycin resistance gene (hpt), and the parental line (Δxt/ft) which has a single integration of the 5'HR region included in the construct (Ruiz- Molina et al, 2021). The MFHR1 transgene was amplified with the same primers used for the qRT-PCR.…”

Section: Nucleic Acids Extraction and Quantitative Pcrmentioning

confidence: 99%

“…MFHR1 is considered a potential therapeutic agent for complement-associated diseases. We produced two variants of this synthetic complement regulator, characterized by an amino acid exchange (V193I), derived from the FH haplotype (V62I) (Ruiz- Molina et al, 2021).…”

Section: A High Copy Number Of Integrated Expression Cassettes Is Important For High Mfhr1 Productivitymentioning

confidence: 99%

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

Ruiz-Molina

Schroeder

et al. 2021

Preprint

Self Cite

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.

“…Among them are the HIV-neutralizing human monoclonal antibody 2G12 produced in Nicotiana tabacum (Ma et al, 2015), the Nicotiana benthamiana-derived virus-like particles as candidate vaccines against influenza, dengue fever or COVID-19, respectively (Ward et al, 2020(Ward et al, , 2021Ponndorf et al, 2021) or α-galactosidase for enzyme replacement therapy in Morbus Fabry treatment produced in the moss Physcomitrella (Shen et al, 2016;Hennermann et al, 2019). Physcomitrella provides several beneficial features for biopharmaceutical production (reviewed in Decker and Reski, 2020): its high rate of somatic homologous recombination enables easy genome engineering (e.g., Strepp et al, 1998;Wiedemann et al, 2018), it is able to produce complex recombinant human proteins and multi-level synthetic complement regulators (Reski et al, 2015;Michelfelder et al, 2017;Top et al, 2019;Ruiz-Molina et al, 2021), can be cultivated under Good Manufacturing Practice (GMP) conditions in suspensions with volumes up to 500 L in photobioreactors (Reski et al, 2018). The promising biopharmaceutical candidates mentioned above demonstrate the potential of plant-based systems in this field.…”

Section: Introductionmentioning

confidence: 99%

Unexpected Arabinosylation after Humanization of Plant Protein N-Glycosylation

Bohlender

Front. Bioeng. Biotechnol.

Hoernstein

et al. 2022

Self Cite

As biopharmaceuticals, recombinant proteins have become indispensable tools in medicine. An increasing demand, not only in quantity but also in diversity, drives the constant development and improvement of production platforms. The N-glycosylation pattern on biopharmaceuticals plays an important role in activity, serum half-life and immunogenicity. Therefore, production platforms with tailored protein N-glycosylation are of great interest. Plant-based systems have already demonstrated their potential to produce pharmaceutically relevant recombinant proteins, although their N-glycan patterns differ from those in humans. Plants have shown great plasticity towards the manipulation of their glycosylation machinery, and some have already been glyco-engineered in order to avoid the attachment of plant-typical, putatively immunogenic sugar residues. This resulted in complex-type N-glycans with a core structure identical to the human one. Compared to humans, plants lack the ability to elongate these N-glycans with β1,4-linked galactoses and terminal sialic acids. However, these modifications, which require the activity of several mammalian enzymes, have already been achieved for Nicotiana benthamiana and the moss Physcomitrella. Here, we present the first step towards sialylation of recombinant glycoproteins in Physcomitrella, human β1,4-linked terminal N-glycan galactosylation, which was achieved by the introduction of a chimeric β1,4-galactosyltransferase (FTGT). This chimeric enzyme consists of the moss α1,4-fucosyltransferase transmembrane domain, fused to the catalytic domain of the human β1,4-galactosyltransferase. Stable FTGT expression led to the desired β1,4-galactosylation. However, additional pentoses of unknown identity were also observed. The nature of these pentoses was subsequently determined by Western blot and enzymatic digestion followed by mass spectrometric analysis and resulted in their identification as α-linked arabinoses. Since a pentosylation of β1,4-galactosylated N-glycans was reported earlier, e.g., on recombinant human erythropoietin produced in glyco-engineered Nicotiana tabacum, this phenomenon is of a more general importance for plant-based production platforms. Arabinoses, which are absent in humans, may prevent the full humanization of plant-derived products. Therefore, the identification of these pentoses as arabinoses is important as it creates the basis for their abolishment to ensure the production of safe biopharmaceuticals in plant-based systems.