Plant-made Pharmaceuticals Research Articles

Plant-made pharmaceuticals

Plant-made pharmaceuticals

Differential prolyl hydroxylation by six Physcomitrella prolyl-4 hydroxylases

Hydroxylation of prolines to 4-trans-hydroxyproline (Hyp) is mediated by prolyl-4 hydroxylases (P4Hs). In plants, Hyps occur in Hydroxyproline-rich glycoproteins (HRGPs), and are frequently O-glycosylated. While both modifications are important, e.g. for cell wall stability, they are undesired in plant-made pharmaceuticals. Sequence motifs for prolyl-hydroxylation were proposed but did not include data from mosses, such as Physcomitrella. We identified six moss P4Hs by phylogenetic reconstruction. Our analysis of 73 Hyps in 24 secretory proteins from multiple mass spectrometry datasets revealed that prolines near other prolines, alanine, serine, threonine and valine were preferentially hydroxylated. About 95 % of Hyps were predictable with combined established methods. In our data, AOV was the most frequent pattern. A combination of 443 AlphaFold models and MS data with 3000 prolines found Hyps mainly on protein surfaces in disordered regions. Moss-produced human erythropoietin (EPO) exhibited O-glycosylation with arabinose chains on two Hyps. This modification was significantly reduced in a p4h1 knock-out (KO) Physcomitrella mutant. Quantitative proteomics with different p4h mutants revealed specific changes in protein amounts, and a modified prolyl-hydroxylation pattern, suggesting a differential function of the Physcomitrella P4Hs. Quantitative RT-PCR revealed a differential effect of single p4h KOs on the expression of the other five p4h genes, suggesting a partial compensation of the mutation. AlphaFold-Multimer models for Physcomitrella P4H1 and its target EPO peptide superposed with the crystal structure of Chlamydomonas P4H1 suggested significant amino acids in the active centre of the enzyme and revealed differences between P4H1 and the other Physcomitrella P4Hs.

Plant-made pharmaceuticals: exploring studies for the production of recombinant protein in plants and assessing challenges ahead.

The production of pharmaceutical compounds in plants is attracting increasing attention, as plant-based systems can be less expensive, safer, and more scalable than mammalian, yeast, bacterial, and insect cell expression systems. Here, we review the history and current status of plant-made pharmaceuticals. Producing pharmaceuticals in plants requires pairing the appropriate plant species with suitable transformation technology. Pharmaceuticals have been produced in tobacco, cereals, legumes, fruits, and vegetables via nuclear transformation, chloroplast transformation, transient expression, and transformation of suspension cell cultures. Despite this wide range of species and methods used, most such efforts have involved the nuclear transformation of tobacco. Tobacco readily generates large amounts of biomass, easily accepts foreign genes, and is amenable to stable gene expression via nuclear transformation. Although vaccines, antibodies, and therapeutic proteins have been produced in plants, such pharmaceuticals are not readily utilized by humans due to differences in glycosylation, and few such compounds have been approved due to a lack of clinical data. In addition, achieving an adequate immune response using plant-made pharmaceuticals can be difficult due to low rates of production compared to other expression systems. Various technologies have recently been developed to help overcome these limitations; however, plant systems are expected to increasingly become widely used expression systems for recombinant protein production.

Seed-Based System for Cost-Effective Production of Vaccine Against Chronic Respiratory Disease in Chickens.

The production of vaccines in plant cells, termed plant-made pharmaceuticals or molecular farming, is a promising technology for scalable production. Compared to mammalian cell lines, like Chinese Hamster Ovary (CHO) or bacterial cells, plants can be grown with less cost on a large scale to make vaccines antigens and therapeutics affordable and accessible worldwide. An innovative application of this alternative system is the production of vaccines in edible tissues that can be consumed orally to deliver protein antigen without any further processing. In this project, we report stable expression of amino acid sequences corresponding to the TM-1 gene of Mycoplasma gallisepticum as a candidate vaccine antigen against Chronic Respiratory Disease (CRD) in chickens using wheat seed′s tissues as a production host. Molecular and immunoblotting analysis confirmed the ubiquitous expression of a recombinant 41.8-kDa protein with an expression level of 1.03 mg/g dry weight in the endosperm tissues. When orally delivered, the plant-made vaccine was effective in terms of developing antibody response in animal model i.e., chicken without any detectable weight loss. Two doses of orally delivered plant-made TM-1 vaccine candidate elicited the immune response and protective effect against MG virus challenge at the level comparable to commercially available inactivated vaccine against CRD. Our study demonstrates that plant-made vaccines are not only safe but also scalable and cost-effective with prolonged stability at room temperature.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12033-022-00554-5.

Unidentified N-glycans by N-glycosidase A were Identified by Nglycosidase F under Denaturing Conditions in Plant Glycoprotein.

The identification of N-glycans in plant glycoproteins or plant-made pharmaceuticals is essential for understanding their structure, function, properties, immunogenicity, and allergenicity (induced by plant-specific core-fucosylation or xylosylation) in the applications of plant food, agriculture, and plant biotechnology. N-glycosidase A is widely used to release the Nglycans of plant glycoproteins because the core-fucosylated N-glycans of plant glycoproteins are hydrolyzed by N-glycosidase A but not by N-glycosidase F. However, the efficiency of Nglycosidase A activity in plant glycoproteins remains unclear. The aim of the study was to elucidate the efficient use of N-glycosidases to identify and quantify the N-glycans of plant glycoproteins; it aimed at identification of released N-glycans by Nglycosidase F and assessment of their relative quantities with a focus on unidentified N-glycans by N-glycosidase A in plant glycoproteins, Phaseolus vulgaris lectin (PHA) and horseradish peroxidase (HRP). Liquid chromatography-tandem mass spectrometry was used to analyze and compare the N-glycans of PHA and HRP treated with either N-glycosidase A or F under denaturing conditions. The relative quantities (%) of each N-glycan (>0.1%) to the total N-glycans (100%) were determined. N-glycosidase A and F released 9 identical N-glycans of PHA, but two additional corefucosylated N-glycans were released by only N-glycosidase A, as expected. By contrast, in HRP, 8 N-glycans comprising 6 core-fucosylated N-glycans, 1 xylosylated N-glycan, and 1 mannosylated N-glycan were released by N-glycosidase A. Moreover, 8 unexpected N-glycans comprising 1 corefucosylated N-glycan, 4 xylosylated N-glycans, and 3 mannosylated N-glycans were released by Nglycosidase F. Of these, 3 xylosylated and 2 mannosylated N-glycans were released by only Nglycansodase F. These results demonstrate that N-glycosidase A alone is insufficient to release the Nglycans of all plant glycoproteins, suggesting that to identify and quantify the released N-glycans of the plant glycoprotein HRP, both N-glycosidase A and F treatments are required.

Compendium on Food Crop Plants as a Platform for Pharmaceutical Protein Production.

Tremendous advances in crop biotechnology related to the availability of molecular tools and methods developed for transformation and regeneration of specific plant species have been observed. As a consequence, the interest in plant molecular farming aimed at producing the desired therapeutic proteins has significantly increased. Since the middle of the 1980s, recombinant pharmaceuticals have transformed the treatment of many serious diseases and nowadays are used in all branches of medicine. The available systems of the synthesis include wild-type or modified mammalian cells, plants or plant cell cultures, insects, yeast, fungi, or bacteria. Undeniable benefits such as well-characterised breeding conditions, safety, and relatively low costs of production make plants an attractive yet competitive platform for biopharmaceutical production. Some of the vegetable plants that have edible tubers, fruits, leaves, or seeds may be desirable as inexpensive bioreactors because these organs can provide edible vaccines and thus omit the purification step of the final product. Some crucial facts in the development of plant-made pharmaceuticals are presented here in brief. Although crop systems do not require more strictly dedicated optimization of methodologies at any stages of the of biopharmaceutical production process, here we recall the complete framework of such a project, along with theoretical background. Thus, a brief review of the advantages and disadvantages of different systems, the principles for the selection of cis elements for the expression cassettes, and available methods of plant transformation, through to the protein recovery and purification stage, are all presented here. We also outline the achievements in the production of biopharmaceuticals in economically important crop plants and provide examples of their clinical trials and commercialization.

Nicotine-free, nontransgenic tobacco (Nicotiana tabacum l.) edited by CRISPR-Cas9.

Nicotine-free, nontransgenic tobacco (Nicotiana tabacum l.) edited by CRISPR-Cas9.

Development of a cucumber green mottle mosaic virus-based expression vector for the production in cucumber of neutralizing epitopes against a devastating animal virus

Virus-based expression systems have been widely exploited for the production of recombinant proteins in plants during the last thirty years. Advances in technology have boosted scale-up manufacturing of plant-made pharmaceuticals to high levels, via the complementation of transient expression and viral vectors. This combination allows proteins of interest to be produced in plants within a matter of days and thus, is well suited for the development of plant-made vaccines or therapeutics against emerging infectious diseases and potential bioterrorism agents. Several plant-based products are currently in varying stages of clinical development. To investigate the viability of virus-based expression systems for plant-made vaccines against porcine reproductive and respiratory syndrome virus (PRRSV), the most devastating threat to the pork industry in Canada, we cloned the full-length genome of a cucumber green mottle mosaic virus (CGMMV) isolate and developed a CGMMV-based expression vector. We further employed this vector to express the neutralizing epitope (NE) of PRRSV glycoprotein 5 (GP5) in cucumber leaves via agroinfiltration. The coding region of the GP5 NE was inserted downstream of the open reading frame for coat protein (CP) and expressed by a readthrough mechanism. The chimeric virus particles were stable and the expression levels reached as high as 35.84 mg/kg of cucumber leaf fresh weight. This study offers a promising solution to the production of a low cost, versatile and robust vaccine for oral administration against PRRSV through a chimeric virus particle display system.

Bulk production of the antiviral lectin griffithsin.

SummaryApplication of plant‐based protein expression systems for bulk production of recombinant protein pharmaceuticals is building momentum. There are considerable regulatory challenges to consider in commercialization of plant‐made pharmaceuticals (PMPs), some of which are inherent to plant‐production systems and others that are common with other production systems, but are new to PMPs because of the youth of the industry. In this review, we discuss our recent and ongoing experience with bulk production of the HIV microbicide candidate, griffithsin (GRFT), utilizing plant‐based transient protein expression, with specific focus on areas relevant to commercial manufacturing of bulk GRFT active pharmaceutical ingredient (API). Analytical programs have been developed for the qualification and monitoring of both the expression vector system and the API detailing our experience and plans for each. Monitoring postpurification protein modifications are discussed in relation to stability and safety programs. Expression, processing and analytics programs are associated with increased manufacturing costs in current good manufacturing practice (cGMP) production because of the required qualification testing. The impact of these costs on the overall cost of goods is particularly relevant to GRFT manufacturing because GRFT, as an HIV microbicide, is most needed in populations at high risk for HIV exposure in resource‐poor countries. Consequently, GRFT for microbicide applications is a very cost‐sensitive recombinant PMP. We have therefore emphasized maintaining a low cost of goods. We provide a review of the literature on the economics of PMPs with various expression systems and how they may impact production costs and complexity.

Current Developments and Future Prospects for Plant-Made Biopharmaceuticals Against Rabies.

Rabies is a prevalent health problem in developing countries. Although vaccines and immunoglobulin treatments are available, their cost and multiple-dose treatments restrict availability. During the last two decades, plants have served as a low-cost platform for biopharmaceuticals production and have been applied to fight against rabies during the last two decades. Herein, I provide a description of the state of the art in the development of plant-made pharmaceuticals against rabies and identify key prospects for the field in terms of novel strategies, immunogen design, and therapeutic antibodies production.

Produção de biológicos em plataformas vegetais e seus impactos na segurança alimentar – análise de lacunas na regulação vigente

The use of genetically modified plants to synthesize proteins that are subsequently processed, regulated, and sold as pharmaceuticals is already a global reality. However, the issue of technological development and industrial production of biological products being expressed in transgenic plants and their impact on the safety of the food chain needs to be discussed more deeply. In this paper, we present an extensive literature review on the standards and regulatory guidelines as well as on biosafety in Brazil with regard to the production of plant-made pharmaceuticals; compare them with the regulatory guidelines of other countries/regions such as USA, Canada, European Union, Cuba, and Argentina; and analyze gaps in the Brazilian regulation. We suggest that some regulations must be reviewed and conclude that the Brazilian regulatory authorities should establish an interrelated regulatory framework together, which would enable industrial production while protecting human and animal health as well as the environment.

Plants: The Future Pharmaceutical Factory

The main objective of this study was to identify prospects, advantages and challenges of plant molecular farming. Plant-made pharmaceuticals (PMPs) and plant made vaccines (PMVs) were intended to be used for human consumption. A broad range of plants, including crops, vegetables and fruits have been investigated for their applicability in production of pharmaceuticals over the last two decades. At present, mainly maize, rice, tobacco and safflower are used in open field production. Contained bioreactor-type systems focus on moss, algae and plant organ culture. Expression systems include stable transformation of plant genomes—both nuclear and plastid, which are also used in the generation of genetically modified crops, plant-viruses and transient expressions. Each expression system (production platform) offers different advantages and disadvantages. Production platform choice is perceptive and depends on a broad range of criterion. This study is based on literature reviews, document analysis and internet database browsing.

A gene responsible for prolyl-hydroxylation of moss-produced recombinant human erythropoietin

Recombinant production of pharmaceutical proteins is crucial, not only for personalized medicine. While most biopharmaceuticals are currently produced in mammalian cell culture, plant-made pharmaceuticals gain momentum. Post-translational modifications in plants are similar to those in humans, however, existing differences may affect quality, safety and efficacy of the products. A frequent modification in higher eukaryotes is prolyl-4-hydroxylase (P4H)-catalysed prolyl-hydroxylation. P4H sequence recognition sites on target proteins differ between humans and plants leading to non-human posttranslational modifications of recombinant human proteins produced in plants. The resulting hydroxyprolines display the anchor for plant-specific O-glycosylation, which bears immunogenic potential for patients. Here we describe the identification of a plant gene responsible for non-human prolyl-hydroxylation of human erythropoietin (hEPO) recombinantly produced in plant (moss) bioreactors. Targeted ablation of this gene abolished undesired prolyl-hydroxylation of hEPO and thus paves the way for plant-made pharmaceuticals humanized via glyco-engineering in moss bioreactors.

Assessment of long-term cryopreservation for production of hCTLA4Ig in transgenic rice cell suspension cultures

For the commercialization of plant-made pharmaceuticals (PMPs) using transgenic plant cell cultures, the establishment of a cell-banking system has been known to be an essential process. Plant cells are traditionally maintained by repeated subcultures. However, this method has several problems including genetic instability of transformed cell lines, time- and cost-consuming. In this study, long-term cryopreserved rice suspension cells were firstly investigated for the production of human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig). The cryopreserved cells for 5 years were regrowed to callus successfully and then suspended into the liquid medium. Consequently, the maximum cell mass and the hCTLA4Ig production were similar levels compared to those of the non-cryopreserved cells (control) even though hCTLA4Ig productivity was 1.7-fold higher than that of control. To further assess the level of improvements in hCTLA4Ig productivity in cryopreserved cells, hCTLA4Ig production profiles were statistically assessed between data of the cryopreserved cells for 5 years and annual data of non-cryopreserved cells maintained by subculture for 5 years. These results also indicate that hCTLA4Ig productivity in cryopreserved cells for 5 years was significantly increased (p-value: <0.001, 95% confidence interval) and it could be related to cell lysis resulting in release of hCTLA4Ig which was confirmed by the measurement of electrolyte leakage. In conclusion, we show that the long-term cryopreservation of transgenic rice cells was possible to support stable cell lines for the production of PMPs.

Production of Plant Made Pharmaceuticals: From Plant Host to Functional Protein

In the last two decades plants have emerged as valuable alternatives to mammalian cells for the production of pharmaceuticals and their potential as expression systems was shown by the commercial availability and acceptance of several plant made therapeuticals in clinical trials. Plants have many advantages over yeast, insect and bacterial expression systems such as the potential to properly fold the expressed proteins and the synthesis of more human-like N-glycans on the proteins. However, several constraints, such as expression yields, downstream processing and structural authenticity, currently limit the widespread use of plant expression systems. In this review, the focus is on the current limitations of plant systems for the production of pharmaceuticals and the possibilities to overcome these obstacles. A comparison is made with insect cell and yeast expression systems. Furthermore, the importance of glycosylation, in particular N-glycosylation for the biological function(s) of therapeutics in the human body will be discussed in detail and an overview of the state of art in the humanization of the N-glycosylation pathway in plants is provided.

Recombinant Cytokines from Plants

Plant-based platforms have been successfully applied for the last two decades for the efficient production of pharmaceutical proteins. The number of commercialized products biomanufactured in plants is, however, rather discouraging. Cytokines are small glycosylated polypeptides used in the treatment of cancer, immune disorders and various other related diseases. Because the clinical use of cytokines is limited by high production costs they are good candidates for plant-made pharmaceuticals. Several research groups explored the possibilities of cost-effective production of animal cytokines in plant systems. This review summarizes recent advances in this field.

Evolution of plant-made pharmaceuticals.

The science and policy of pharmaceuticals produced and/or delivered by plants has evolved over the past twenty-one years from a backyard remedy to regulated, purified products. After seemingly frozen at Phase I human clinical trials with six orally delivered plant-made vaccines not progressing past this stage over seven years, plant-made pharmaceuticals have made a breakthrough with several purified plant-based products advancing to Phase II trials and beyond. Though fraught with the usual difficulties of pharmaceutical development, pharmaceuticals made by plants have achieved pertinent milestones albeit slowly compared to other pharmaceutical production systems and are now at the cusp of reaching the consumer. Though the current economic climate begs for cautious investment as opposed to trail blazing, it is perhaps a good time to look to the future of plant-made pharmaceutical technology to assist in planning for future developments in order not to slow this technology’s momentum. To encourage continued progress, we highlight the advances made so far by this technology, particularly the change in paradigms, comparing developmental timelines, and summarizing the current status and future possibilities of plant-made pharmaceuticals.

Use of uniconazole to control plant height for an industrial/pharmaceutical maize platform

A variety of crops and their production systems have been proposed as platforms for plant-made pharmaceuticals (PMPs) and plant-made industrial compounds (PMICs), each with their own advantages and challenges. Maize grown in isolated, confined environments avoids several challenging issues associated with future production of PMPs/PMICs. When grown under optimizing conditions of temperature, nutrition, and water status, however, maize tends to elongate excessively, which contributes to lodging and interferes with delivery of sufficient overhead electric lighting to the inner foliar canopy where ears develop. Suppression of extensive stem elongation without compromising grain yield would help in delivering overhead light into the foliar canopy for efficient grain production. The plant-growth retardant uniconazole (UCZ) was tested for its effects on maize height and grain yield. UCZ reduced plant height effectively, but dosages that strongly suppressed stem elongation also decreased grain yield, ear length, and cob weight. Duration of growth suppression was prolonged at high UCZ concentrations. UCZ also induced branching of maize, which was enhanced as concentrations increased. A range of ear types occurred on branches, with some conversion of ears to tassels at high UCZ concentrations. Minimum stem diameter declined with increasing UCZ concentration. Plant height was reduced from 306 cm to 232 cm (−25%) using a concentration of 1.0 mg/L UCZ without affecting grain yield of 180–200 g per plant. This low UCZ concentration caused 1–2 basal branches to form that would not affect planting density in a cropping situation.

New areas of plant-made pharmaceuticals

On October 15 2010 the meeting ‘Recombinant Pharmaceutical Manufacturing from Plants – The Future of Molecular Farming’ hosted by EuroScicon was held at BioPark Hertfordshire, Welwyn Garden city, UK. The scientific program of this very eventful meeting was wide ranging and covered diverse aspects of biopharming. The highlights presented included: safety issues in biopharming; coexpression of multiple proteins; steps towards vaccine generation; and engineering of secondary metabolites and medicinal plants. This article summarizes the stimulating scientific presentations and fruitful panel discussions that subsequently arose during and after this event.

The economic potential of plant-made pharmaceuticals in the manufacture of biologic pharmaceuticals

Biologic pharmaceuticals have emerged to treat a number of diseases such as cancer and immune disorders. Although these drugs provide significant benefits, they have tended to be costly to produce. Production technologies that grow biologic drugs in plants offer the potential to lower those costs. This article uses a system of simulation models to compare the economics of plant made pharmaceuticals (PMP) to incumbent productions systems. Although it is found that significant cost savings are possible, RD investment in PMPs continue to be limited, which suggests that there are a number of obstacles that limit adoption.