植物生物反应器生产药物蛋白的前景

转基因植物作为生物反应器生产重要的药用蛋白,如抗体、血液替代品和疫苗,为健康保健和科学研究提供充足的生物药物,满足人们日益增长的需要。本文综述了这一领域的研究进展以及商业化前景。     

Gene amplification and vector engineering to achieve rapid and high-level therapeutic protein production using the Dhfr-based CHO cell selection system

Demand is increasing for therapeutic biopharmaceuticals such as monoclonal antibodies. Achieving maximum production of these recombinant proteins under developmental time constraints has been a recent focus of study. The majority of these drugs are currently produced in altered Chinese hamster ovary (CHO) cells due to the high viability and the high densities achieved by these cells in suspension cultures. However, shortening the process of developing and isolating high-producing cell lines remains a challenge. This article focuses on current expression systems used to produce biopharmaceuticals in CHO cells and current methods being investigated to produce biopharmaceuticals more efficiently. The methods discussed include modified gene amplification methods, modifying vectors to improve expression of the therapeutic gene and improving the method of selecting for high-producing cells. Recent developments that use gene targeting as a method for increasing production are discussed.     

Glycoengineering in plants for the development of N-glycan structures compatible with biopharmaceuticals

Plants and plant cells are emerging as promising alternatives for biopharmaceutical production with improved safety and efficiency. Plant cells are capable of performing post-translational modifications (PTMs) similar to those of mammalian cells and are safer than mammalian cells with regard to contamination by infectious pathogens, including animal viruses. However, a major obstacle to producing biopharmaceuticals in plants lies in the fact that plant-derived N-glycans include plant-specific sugar residues such as β1,2-xylose and α1,3-fucose attached to a pentasaccharide core (Man₃GlcNAc₂) as well as β1,3-galactose and α1,4-fucose involved in Lewis a (Le^a) epitope formation that can evoke allergic responses in the human body. In addition, sugar residues such as α1,6-fucose, β1,4-galactose and α2,6-sialic acid, which are thought to play important roles in the activity, transport, delivery and half-life of biopharmaceuticals are absent among the N-glycans naturally found in plants. In order to take advantage of plant cells as a system in which to produce biopharmaceuticals development of plants producing N-glycan structures compatible with biopharmaceuticals is necessary. In this article we summarize the current state of biopharmaceutical production using plants as well as what is known about N-glycosylation processes occurring in the endoplasmic reticulum and Golgi apparatus in plants. Finally, we propose and discuss a strategy for and the associated technical barriers of producing customized N-glycans via removal of enzyme genes that add plant-specific sugar residues and introducing enzyme genes that add sugar residues absent in plants.     

Recent developments in the use of transgenic plants for the production of human therapeutics and biopharmaceuticals

In recent years there has been a dramatic increase in the application of plant biotechnology for the production of a variety of commercially valuable simple and complex biological molecules (biologics) for use in human and animal healthcare. Transgenic whole plants and plant cell culture systems have been developed that have the capacity to economically produce large-scale quantities of antibodies and antibody fragments, antigens and/or vaccine epitopes, metabolic enzymes, hormones, (neuro)peptides and a variety of biologically active complexes and secondary metabolites for direct use as therapeutic agents or diagnostic tools in the medical healthcare industry. As the products of genetically modified plants make their way from concept to commercialization the associated risks and acceptance by the public has been become a focal point. In this paper, we summarize the recent advances made in the use of transgenic plants and plant cell cultures as biological factories for the production of human therapeutics and biopharmaceuticals and discuss the long-term potential of `molecular farming' as a low-cost, efficient method for the production of biological materials with demonstrated utility to the pharmaceutical industry or medical community.     

The production of biopharmaceuticals in plant systems

Biopharmaceuticals present the fastest growing segment in the pharmaceutical industry, with an ever widening scope of applications. Whole plants as well as contained plant cell culture systems are being explored for their potential as cheap, safe, and scalable production hosts. The first plant-derived biopharmaceuticals have now reached the clinic. Many biopharmaceuticals are glycoproteins; as the Golgi N-glycosylation machinery of plants differs from the mammalian machinery, the N-glycoforms introduced on plant-produced proteins need to be taken into consideration. Potent systems have been developed to change the plant N-glycoforms to a desired or even superior form compared to the native mammalian N-glycoforms. This review describes the current status of biopharmaceutical production in plants for industrial applications. The recent advances and tools which have been utilized to generate glycoengineered plants are also summarized and compared with the relevant mammalian systems whenever applicable.     

Plant-derived pharmaceuticals for the developing world

Plant-produced vaccines and therapeutic agents offer enormous potential for providing relief to developing countries by reducing the incidence of infant mortality caused by infectious diseases. Vaccines derived from plants have been demonstrated to effectively elicit an immune response. Biopharmaceuticals produced in plants are inexpensive to produce, require fewer expensive purification steps, and can be stored at ambient temperatures for prolonged periods of time. As a result, plant-produced biopharmaceuticals have the potential to be more accessible to the rural poor. This review describes current progress with respect to plant-produced biopharmaceuticals, with a particular emphasis on those that target developing countries. Specific emphasis is given to recent research on the production of plant-produced vaccines toward human immunodeficiency virus, malaria, tuberculosis, hepatitis B virus, Ebola virus, human papillomavirus, rabies virus and common diarrheal diseases. Production platforms used to express vaccines in plants, including nuclear and chloroplast transformation, and the use of viral expression vectors, are described in this review. The review concludes by outlining the next steps for plant-produced vaccines to achieve their goal of providing safe, efficacious and inexpensive vaccines to the developing world.     

Bioreactor technology: a novel industrial tool for high-tech production of bioactive molecules and biopharmaceuticals from plant roots

Plants are the richest source for different bioactive molecules. Because of the vast number of side effects associated with synthetic pharmaceuticals, medical biotechnologists turned to nature to provide new promising therapeutic molecules from plant biofactories. The large-scale availability of the disease- and pesticide-free raw material is, however, restricted in vivo. Many bioactive plant secondary metabolites are accumulated in roots. Engineered plants can also produce human therapeutic proteins. Vaccines and diagnostic monoclonal antibodies can be won from their roots, so that engineered plants hold immense potential for the biopharmaceutical industry. To obtain sufficient amounts of the plant bioactive molecules for application in human therapy, adventitious and hairy roots have to be cultured in in vitro systems. High-tech pilot-scale bioreactor technology for the establishment of a long-term adventitious root culture from biopharmaceutical plants has recently been established. In this review, I briefly discuss a technology for cultivating bioactive molecule-rich adventitious and hairy roots from plants using a high-tech bioreactor system, as well as the principles and application of genome-restructuring mechanisms for plant-based biopharmaceutical production from roots. High-tech bioreactor-derived bioactive phytomolecules and biopharmaceuticals hold the prospect of providing permanent remedies for improving human well-being.     

Hightech aus der grünen Apotheke. Biopharmazeutika aus Pflanzen

Hightech from Natures Pharmacy Plants produce a plethora of valuable natural products, many of which are used as pharmaceuticals. Today, a large fraction of the novel pharmaceuticals entering the market are biomolecules of proteinaceous nature (antibodies, hormones, cytokines, vaccines) and they are produced in transgenic organisms like bacteria, yeast, or mammalian cell cultures. Plants are also capable to serve as a production host for novel therapeutics like monoclonal antibodies, hormones like insulin, or subunit vaccines. Transgenic plants and plant cell cultures are already modified to produce protein biopharmaceuticals and vaccines on a large scale basis and in some aspects they have clear advantages over conventional production hosts (e.g. cost of goods, speed of production, or posttranslational modification of therapeutic proteins). Therefore, plant biotechnology could create entirely novel medicinal plants with applications not known before.     

Genetic engineering approaches to improve posttranslational modification of biopharmaceuticals in different production platforms

The number of approved biopharmaceuticals, where product quality attributes remain of major importance, is increasing steadily. Within the available variety of expression hosts, the production of biopharmaceuticals faces diverse limitations with respect to posttranslational modifications (PTM), while different biopharmaceuticals demand different forms and specifications of PTMs for proper functionality. With the growing toolbox of genetic engineering technologies, it is now possible to address general as well as host- or biopharmaceutical-specific product quality obstacles. In this review, we present diverse expression systems derived from mammalians, bacteria, yeast, plants, and insects as well as available genetic engineering tools. We focus on genes for knockout/knockdown and overexpression for meaningful approaches to improve biopharmaceutical PTMs and discuss their applicability as well as future trends in the field.     

Expression of a recombinant human erythropoietin in suspension cell cultures of Arabidopsis, tobacco and Medicago

In the last two decades, the use of plants to produce recombinant proteins and particularly biopharmaceuticals has become an attractive alternative to established systems. This is due to advantages in scalability, economy and safety. In addition the expression of recombinant proteins in plants can also be achieved utilizing in vitro cell suspensions with all the advantages such systems confer, such as product consistency, production ??on demand?? and the ability to perform the entire process according to good manufacturing practices. In this study we have produced the glycosylated human hormone Erythropoietin (EPO), in Medicago truncatula and Arabidopsis thaliana plants and also in cultured cell lines of tobacco, Medicago and Arabidopsis. We have also tested two different versions of the protein, one with a KDEL tag for targeted expression in the Endoplasmic Reticulum, and an untagged version expected to be secreted to the apoplast. The recombinant protein was detected in the plant leaf extracts and in the cultured cell lines. In the latter, the rEPO was detected in the cell extracts and in the spent culture medium. It was possible to recover the KDEL version of rEPO from crude cell extracts by nickel affinity chromatography, however the secreted form did not bind to the Ni- agarose beads which may indicate a possible internalization of the his-tag in the folded protein. Although the yield of rEPO obtained in cell suspensions was not as high as expected, the protein was successfully produced and secreted into the culture medium, reinforcing that plant cell suspension cultures are a promising system for production of human biopharmaceuticals.     

The emerging role of mass spectrometry-based proteomics in molecular pharming practices

Molecular pharming relies on the integration of foreign genes into a plant system for production of the desired recombinant protein. The speed, scalability, and lack of contaminating human pathogens highlights plants as an enticing and feasible system to produce diverse protein-based products, including vaccines, antibodies, and enzymes. However, limitations of expression levels, host defense responses, and production irregularities underscore distinct areas for improvement within the molecular pharming pipeline. Within the past five years, mass spectrometry-based proteomics has begun to address these critical areas and show promise in advancing our understanding of the complex biological systems driving molecular pharming. Further, opportunities to leverage comprehensive proteome profiling have surfaced to meet good manufacturing practice regulations and move biopharmaceuticals derived from plants into mainstream production.     

Transgenic plants as factories for biopharmaceuticals

Plants have considerable potential for the production of biopharmaceutical proteins and peptides because they are easily transformed and provide a cheap source of protein. Several biotechnology companies are now actively developing, field testing, and patenting plant expression systems, while clinical trials are proceeding on the first biopharmaceuticals derived from them. One transgenic plant-derived biopharmaceutical, hirudin, is now being commercially produced in Canada for the first time. Product purification is potentially an expensive process, and various methods are currently being developed to overcome this problem, including oleosin-fusion technology, which allows extraction with oil bodies. In some cases, delivery of a biopharmaceutical product by direct ingestion of the modified plant potentially removes the need for purification. Such biopharmaceuticals and edible vaccines can be stored and distributed as seeds, tubers, or fruits, making immunization programs in developing countries cheaper and potentially easier to administer. Some of the most expensive biopharmaceuticals of restricted availability, such as glucocerebrosidase, could become much cheaper and more plentiful through production in transgenic plants.     

Towards high-yield production of pharmaceutical proteins with plant cell suspension cultures

“Molecular farming” in plants with significant advantages in cost and safety is touted as a promising platform for the production of complex pharmaceutical proteins. While whole-plant produced biopharmaceuticals account for a significant portion of the preclinical and clinical pipeline, plant cell suspension culture, which integrates the merits of whole-plant systems with those of microbial fermentation, is emerging as a more compliant alternative “factory”. However, low protein productivity remains a major obstacle that limits extensive commercialization of plant cell bioproduction platform. This review highlights the advantages and recent progress in plant cell culture technology and outlines viable strategies at both the biological and process engineering levels for advancing the economic feasibility of plant cell-based protein production. Approaches to overcome and solve the associated challenges of this culture system that include non-mammalian glycosylation and genetic instability will also be discussed.     

Advances in chloroplast engineering

The chloroplast is a pivotal organelle in plant cells and eukaryotic algae to carry out photosynthesis, which provides the primary source of the world＇s food. The expression of foreign genes in chloroplasts offers several advantages over their expression in the nucleus： high-level expression, transgene stacking in operons and a lack of epigenetic interference allowing stable transgene expression. In addition, transgenic chloroplasts are generally not transmitted through pollen grains because of the cytoplasmic localization. In the past two decades, great progress in chloroplast engineering has been made. In this paper, we review and highlight recent studies of chloroplast engineering, including chloroplast transformation procedures, controlled expression of plastid transgenes in plants, the expression of foreign genes for improvement of plant traits, the production of biopharmaceuticals, metabolic pathway engineering in plants, plastid transformation to study RNA editing, and marker gene excision system.     

Green biofactories: recombinant protein production in plants

Until recently, low accumulation levels have been the major bottleneck for plant-made recombinant protein production. However, several breakthroughs have been described in the past few years allowing for very high accumulation levels, mainly through chloroplast transformation and transient expression, coupled with subcellular targeting and protein fusions. Another important factor influencing our ability to use plants for the production of recombinant proteins is the availability of quick and simple purification strategies. Recent developments using oleosin, zein, ELP and hydrophobin fusion tags have shown promise as efficient and cost-effective methods for non-chromatographic separation. Furthermore, plant glycosylation is a major barrier to the parenteral administration of plant-made biopharmaceuticals because of potential immunogenicity concerns. A major effort has been invested in humanizing plant glycosylation, and several groups have been able to reduce or eliminate immunogenic glycans while introducing mammalian-specific glycans. Finally, biosafety issues and public perception are essential for the acceptance of plants as bioreactors for the production of proteins. Over recent years, it has become clear that food and feed plants carry an inherent risk of contaminating our food supply, and thus much effort has focused on the use of non-food plants. Presently, Nicotiana benthamiana has emerged as the preferred host for transient expression, while tobacco is most frequently used for chloroplast transformation. In this review, we focus on the main issues hindering the economical production of recombinant proteins in plants, describing the current efforts for addressing these limitations, and we include an extensive list of recent patents generated with the intention of solving these limitations.     

Leveraging mathematical models for optimizing filter utility at manufacturing scale

In the production of biopharmaceuticals depth filters followed by sterile filters are often employed to remove residual cell debris present in the feed stream. In the back drop of a global pandemic, supply chains associated with the production of biopharmaceuticals have been constrained. These constraints have limited the available amount of depth filters for the manufacture of biologics. This has placed manufacturing facilities in a difficult position having to choose between running processes with reduced number of depth filters and risking a failed batch or the prospect of plants going into temporary shutdown until the depth filter resources are replenished. This communication describes a modeling based method that leverages manufacturing scale filtration data to predict the depth filter performance with a reduced number of filters and an increased operational flux. This method can be used to quantify the acceptable level of area reduction before which the filtration process performance is affected. This enables facilities to manage their filter inventory avoiding potential plant shutdowns and reduces the risks of negative depth filter performance.     

Recombinant protein production and streptomycetes

The biopharmaceutical market has come a long way since 1982, when the first biopharmaceutical product, recombinant human insulin, was launched. Just over 200 biopharma products have already gained approval. The global market for biopharmaceuticals which is currently valued at over US$99 billion has been growing at an impressive compound annual growth rate over the previous years. To produce these biopharmaceuticals and other industrially important heterologous proteins, different prokaryotic and eukaryotic expression systems are used. All expression systems have some advantages as well as some disadvantages that should be considered in selecting which one to use. Choosing the best one requires evaluating the options--from yield to glycosylation, to proper folding, to economics of scale-up. No host cell from which all the proteins can be universally expressed in large quantities has been found so far. Therefore, it is important to provide a variety of host-vector expression systems in order to increase the opportunities to screen for the most suitable expression conditions or host cell. In this overview, we focus on Streptomyces lividans, a Gram-positive bacterium with a proven excellence in secretion capacity, as host for heterologous protein production. We will discuss its advantages and disadvantages, and how with systems biology approaches strains can be developed to better producing cell factories.     

In vitro protein refolding by chromatographic procedures

In vitro protein refolding is still a bottleneck in both structural biology and in the development of new biopharmaceuticals, especially for commercially important polypeptides that are overexpressed in Escherichia coli. This review focuses on protein refolding methods based on column procedures because recent advances in chromatographic refolding have shown promising results.     

Animal cell cultures: recent achievements and perspectives in the production of biopharmaceuticals

There has been a rapid increase in the number and demand for approved biopharmaceuticals produced from animal cell culture processes over the last few years. In part, this has been due to the efficacy of several humanized monoclonal antibodies that are required at large doses for therapeutic use. There have also been several identifiable advances in animal cell technology that has enabled efficient biomanufacture of these products. Gene vector systems allow high specific protein expression and some minimize the undesirable process of gene silencing that may occur in prolonged culture. Characterization of cellular metabolism and physiology has enabled the design of fed-batch and perfusion bioreactor processes that has allowed a significant improvement in product yield, some of which are now approaching 5 g/L. Many of these processes are now being designed in serum-free and animal-component-free media to ensure that products are not contaminated with the adventitious agents found in bovine serum. There are several areas that can be identified that could lead to further improvement in cell culture systems. This includes the down-regulation of apoptosis to enable prolonged cell survival under potentially adverse conditions. The characterization of the critical parameters of glycosylation should enable process control to reduce the heterogeneity of glycoforms so that production processes are consistent. Further improvement may also be made by the identification of glycoforms with enhanced biological activity to enhance clinical efficacy. The ability to produce the ever-increasing number of biopharmaceuticals by animal cell culture is dependent on sufficient bioreactor capacity in the industry. A recent shortfall in available worldwide culture capacity has encouraged commercial activity in contract manufacturing operations. However, some analysts indicate that this still may not be enough and that future manufacturing demand may exceed production capacity as the number of approved biotherapeutics increases.