Production Of Human Therapeutic Proteins Research Articles

Genetically Engineered Pigs as Efficient Salivary Gland Bioreactors for Production of Therapeutically Valuable Human Nerve Growth Factor.

Farm animal salivary glands hold great potential as efficient bioreactors for production of human therapeutic proteins. Nerve growth factor (NGF) is naturally expressed in animal salivary glands and has been approved for human clinical treatment. This study aims to employ transgenic (TG) pig salivary gland as bioreactors for efficient synthesis of human NGF (hNGF). hNGF-TG pigs were generated by cloning in combination with piggyBac transposon-mediated gene transfer. These hNGF-TG pigs specifically expressed hNGF protein in their salivary glands and secreted it at high levels into saliva. Surgical and nonsurgical approaches were developed to efficiently collect saliva from hNGF-TG pigs. hNGF protein was successfully purified from collected saliva and was verified to be biologically active. In an additional step, the double-transgenic pigs, where the endogenous porcine NGF (pNGF) gene was replaced by another copy of hNGF transgene, were created by cloning combined with CRISPR/Cas9-mediated homologous recombination. These double-transgenic pigs expressed hNGF but not pNGF, thus avoiding possible “contamination” of hNGF with pNGF protein during purification. In conclusion, TG pig salivary glands can be used as robust bioreactors for a large-scale synthesis of functional hNGF or other valuable proteins. This new animal pharming method will benefit both human health and biomedicine.

Production of transgenic chickens constitutively expressing human erythropoietin (hEPO): Problems with uncontrollable overexpression of hEPO gene

The chicken is a promising candidate as a bioreactor for the economical mass production of human therapeutic proteins. Here, we report the successful generation of transgenic chickens that produce high concentrations of human erythropoietin (hEPO) in the blood. Using a Moloney murine leukemia virus (MoMLV)-based pseudotyped retrovirus vector packaged with vesicular stomatitis virus G glycoprotein (VSV-G), the hEPO gene under the control of cytomegalovirus (CMV) promoter was introduced to the blastoderm of freshly laid chicken eggs (stage X). Out of 200 injected eggs, 12 chicks were hatched after 21 days of incubation, and all of the G0 hatched chicks expressed the vector-encoded hEPO gene. One of the G0 roosters successfully transmitted the hEPO gene to its G1 progeny by crossing with non-transgenic hens. The concentration of hEPO protein in the chicken blood serum was as high as 90 μg/mL. Although humans and chickens belong to different classes of the phylogenetic tree, human EPO caused devastating problems in transgenic chickens, including sudden death, polycythemia, vasodilation, and so on, which may be due to the uncontrolled constitutive expression of exogenous protein in the chicken body. Despite many disorders, however, we were able to generate chicks of G2 generation sired by a rooster of G1 generation confirming successful establishment of a new line of transgenic chicken characterized by high expression of the hEPO gene. With these chickens, we believe that studies on the evaluating the possibilities of the transgenic animal-mediated bio-pharming and on the hEPO-induced physiological side effects will be greatly facilitated.

Vaccination via Chloroplast Genetics: Affordable Protein Drugs for the Prevention and Treatment of Inherited or Infectious Human Diseases.

Plastid-made biopharmaceuticals treat major metabolic or genetic disorders, including Alzheimer's, diabetes, hypertension, hemophilia, and retinopathy. Booster vaccines made in chloroplasts prevent global infectious diseases, such as tuberculosis, malaria, cholera, and polio, and biological threats, such as anthrax and plague. Recent advances in this field include commercial-scale production of human therapeutic proteins in FDA-approved cGMP facilities, development of tags to deliver protein drugs to targeted human cells or tissues, methods to deliver precise doses, and long-term stability of protein drugs at ambient temperature, maintaining their efficacy. Codon optimization utilizing valuable information from sequenced chloroplast genomes enhanced expression of eukaryotic human or viral genes in chloroplasts and offered unique insights into translation in chloroplasts. Support from major biopharmaceutical companies, development of hydroponic production systems, and evaluation by regulatory agencies, including the CDC, FDA, and USDA, augur well for advancing this novel concept to the clinic and revolutionizing affordable healthcare.

Genome-scale metabolic model of Pichia pastoris with native and humanized glycosylation of recombinant proteins.

Pichia pastoris is used for commercial production of human therapeutic proteins, and genome-scale models of P. pastoris metabolism have been generated in the past to study the metabolism and associated protein production by this yeast. A major challenge with clinical usage of recombinant proteins produced by P. pastoris is the difference in N-glycosylation of proteins produced by humans and this yeast. However, through metabolic engineering, a P. pastoris strain capable of producing humanized N-glycosylated proteins was constructed. The current genome-scale models of P. pastoris do not address native nor humanized N-glycosylation, and we therefore developed ihGlycopastoris, an extension to the iLC915 model with both native and humanized N-glycosylation for recombinant protein production, but also an estimation of N-glycosylation of P. pastoris native proteins. This new model gives a better prediction of protein yield, demonstrates the effect of the different types of N-glycosylation of protein yield, and can be used to predict potential targets for strain improvement. The model represents a step towards a more complete description of protein production in P. pastoris, which is required for using these models to understand and optimize protein production processes.

Recombinant glycoprotein production in human cell lines.

The most important properties of a protein are determined by its primary structure, its amino acid sequence. However, protein features can be also modified by a large number of posttranslational modifications. These modifications can occur during or after the synthesis process, and glycosylation appears as the most common posttranslational modification. It is estimated that 50% of human proteins have some kind of glycosylation, which has a key role in maintaining the structure, stability, and function of the protein. Besides, glycostructures can also influence the pharmacokinetics and immunogenicity of the protein. Although the glycosylation process is a conserved mechanism that occurs in yeast, plants, and animals, several studies have demonstrated significant differences in the glycosylation pattern in recombinant proteins expressed in mammalian, yeast, and insect cells. Thus, currently, important efforts are being done to improve the systems for the expression of recombinant glycosylated proteins. Among the different mammalian cell lines used for the production of recombinant proteins, a significant difference in the glycosylation pattern that can alter the production and/or activity of the protein exists. In this context, human cell lines have emerged as a new alternative for the production of human therapeutic proteins, since they are able to produce recombinant proteins with posttranslational modifications similar to its natural counterpart and reduce potential immunogenic reactions against nonhuman epitopes. This chapter describes the steps necessary to produce a recombinant glycoprotein in a human cell line in small scale and also in bioreactors.

MALDI‐TOF characterization of hGH1 produced by hairy root cultures of Brassica oleracea var. italica grown in an airlift with mesh bioreactor

Expression systems based on plant cells, tissue, and organ cultures have been investigated as an alternative for production of human therapeutic proteins in bioreactors. In this work, hairy root cultures of Brassica oleracea var. italica (broccoli) were established in an airlift with mesh bioreactor to produce isoform 1 of the human growth hormone (hGH1) as a model therapeutic protein. The hGH1 cDNA was cloned into the pCAMBIA1105.1 binary vector to induce hairy roots in hypocotyls of broccoli plantlets via Agrobacterium rhizogenes. Most of the infected plantlets (90%) developed hairy roots when inoculated before the appearance of true leaves, and keeping the emerging roots attached to hypocotyl explants during transfer to solid Schenk and Hildebrandt medium. The incorporation of the cDNA into the hairy root genome was confirmed by PCR amplification from genomic DNA. The expression and structure of the transgenic hGH1 was assessed by ELISA, western blot, and MALDITOF-MS analysis of the purified protein extracted from the biomass of hairy roots cultivated in bioreactor for 24 days. Production of hGH1 was 5.1 ± 0.42 µg/g dry weight (DW) for flask cultures, and 7.8 ± 0.3 µg/g DW for bioreactor, with productivity of 0.68 ± 0.05 and 1.5 ± 0.06 µg/g DW*days, respectively, indicating that the production of hGH1 was not affected by the growth rate, but might be affected by the culture system. These results demonstrate that hairy root cultures of broccoli have potential as an alternative expression system for production of hGH1, and might also be useful for production of other therapeutic proteins.

Expression and Production of Therapeutic Recombinant Human Platelet-Derived Growth Factor-BB in Pleurotus eryngii

Recombinant human platelet-derived growth factor-BB (rhPDGF-BB) is widely used in many therapeutic applications. Until now, there has been no report on rhPDGF-BB expressed in fungi. In this study, we tested whether Pleurotus eryngii could support the expression of human therapeutic rhPDGF-BB protein. A binary vector pCAMBIA1304 containing the hPDGF-BB gene was constructed and introduced into P. eryngii via Agrobacterium tumefaciens-mediated transformation. The transformation of hPDGF-BB gene was confirmed by Southern blot and PCR, whereas the expression was confirmed by Western blot analysis. The recombinant hPDGF-BB reached a maximum expression level of 1.98% of total soluble protein in transgenic mycelia and was in dimeric form. A bioassay revealed that hPDGF-BB expressed in P. eryngii increased proliferation of NIH-3T3 cells similarly to standard material. These results suggest that P. eryngii can be a robust system for the production of human therapeutic proteins including the hPDGF-BB.

Production of biologically active human thioredoxin 1 protein in lettuce chloroplasts.

The production of human therapeutic proteins in plants provides opportunities for low-cost production, and minimizes the risk of contamination from potential human pathogens. Chloroplast genetic engineering is a particularly promising strategy, because plant chloroplasts can produce large amounts of foreign target proteins. Oxidative stress is a key factor in various human diseases. Human thioredoxin 1 (hTrx1) is a stress-induced protein that functions as an antioxidant against oxidative stress, and overexpression of hTrx1 has been shown to suppress various diseases in mice. Therefore, hTrx1 is a prospective candidate as a new human therapeutic protein. We created transplastomic lettuce expressing hTrx1 under the control of the psbA promoter. Transplastomic plants grew normally and were fertile. The hTrx1 protein accumulated to approximately 1% of total soluble protein in mature leaves. The hTrx1 protein purified from lettuce leaves was functionally active, and reduced insulin disulfides. The purified protein protected mouse insulinoma line 6 cells from damage by hydrogen peroxide, as reported previously for a recombinant hTrx1 expressed in Escherichia coli. This is the first report of expression of the biologically active hTrx1 protein in plant chloroplasts. This research opens up possibilities for plant-based production of hTrx1. Considering that this expression host is an edible crop plant, this transplastomic lettuce may be suitable for oral delivery of hTrx1.

The microalga Chlamydomonas reinhardtii as a platform for the production of human protein therapeutics

Microalgae are a diverse group of eukaryotic photosynthetic microorganisms. While microalgae play a crucial role in global carbon fixation and oxygen evolution, these organisms have recently gained much attention for their potential role in biotechnological and industrial applications, such as the production of biofuels. We investigated the potential of the microalga Chlamydomonas reinhardtii to be a platform for the production of human therapeutic proteins. C. reinhardtii is a unicellular freshwater green alga that has served as a popular model alga for physiological, molecular, biochemical and genetic studies. As such, the molecular toolkit for this microorganism is highly developed, including well-established methods for genetic transformation and recombinant gene expression. We transformed the chloroplast genome of C. reinhardtii with seven unrelated genes encoding for current or potential human therapeutic proteins and found that four of these genes supported protein accumulation to levels that are sufficient for commercial production. Furthermore, the algal-produced proteins were bioactive. Thus, the microalga C. reinhardtii has the potential to be a robust platform for human therapeutic protein production.

DNA spike studies for demonstrating improved clearance on chromatographic media

DNA spike clearance methods were used to demonstrate improved clearance factors on anion exchange and hydrophobic interaction columns used in the production of human therapeutic proteins. DNA clearance at large-scale was first measured for a monoclonal antibody expressed in Chinese Hamster Ovary (CHO) cells and an antibody fragment expressed in Escherichia coli. Small-scale spike experiments were then performed on individual chromatographic steps using host-specific DNA paired with TaqMan PCR assay methods. This approach has advantages of improved specificity, sensitivity, cost and throughput compared to other types of spike clearance methods. The anion exchange column used in the monoclonal antibody process was shown to have very high capacity for CHO DNA, resulting in greater than 7.1 log reduction. The anion exchange and hydrophobic interaction columns used in the antibody fragment process were shown to have high E. coli DNA clearance capability, with greater than 5.1 and 5.3 logs clearance, respectively. Compared to the large-scale process, higher log reduction values were achieved in small-scale spike clearance studies by challenging the chromatographic steps with load DNA levels 2–5 logs higher than the large-scale process levels. Using highly specific and sensitive spike clearance methods, we demonstrated consistently high DNA clearance factors for each of the production processes that meet industry and regulatory standards for human therapeutics. The method is applicable to a broad range of industrial scale processes where demonstration of the robustness of DNA clearance is necessary to support development or licensure of biopharmaceutical products.

Accumulation and processing of a recombinant protein designed as a cleavable fusion to the endogenous Rubisco LSU protein in Chlamydomonas chloroplast

BackgroundExpression of recombinant proteins in green algal chloroplast holds substantial promise as a platform for the production of human therapeutic proteins. A number of proteins have been expressed in the chloroplast of Chlamydomonas reinhardtii, including complex mammalian proteins, but many of these proteins accumulate to significantly lower levels than do endogenous chloroplast proteins. We examined if recombinant protein accumulation could be enhanced by genetically fusing the recombinant reporter protein, luciferase, to the carboxy-terminal end of an abundant endogenous protein, the large subunit of ribulose bisphosphate carboxylase (Rubisco LSU). Additionally, as recombinant proteins fused to endogenous proteins are of little clinical or commercial value, we explored the possibility of engineering our recombinant protein to be cleavable from the endogenous protein in vivo. This strategy would obviate the need for further in vitro processing steps in order to produce the desired recombinant protein. To achieve this, a native protein-processing site from preferredoxin (preFd) was placed between the Rubisco LSU and luciferase coding regions in the fusion protein construct.ResultsThe luciferase from the fusion protein accumulated to significantly higher levels than luciferase expressed alone. By eliminating the endogenous Rubisco large subunit gene (rbcL), we achieved a further increase in luciferase accumulation with respect to luciferase expression in the WT background. Importantly, near-wild type levels of functional Rubisco holoenzyme were generated following the proteolytic removal of the fused luciferase, while luciferase activity for the fusion protein was almost ~33 times greater than luciferase expressed alone. These data demonstrate the utility of using fusion proteins to enhance recombinant protein accumulation in algal chloroplasts, and also show that engineered proteolytic processing sites can be used to liberate the exogenous protein from the endogenous fusion partner, allowing for the purification of the intended mature protein.ConclusionThese results demonstrate the utility of fusion proteins in algal chloroplast as a method to increase accumulation of recombinant proteins that are difficult to express. Since Rubisco is ubiquitous to land plants and green algae, this strategy may also be applied to higher plant transgenic expression systems.

High‐yield production of authentic human growth hormone using a plant virus‐based expression system

We describe here a high-yield transient expression system for the production of human growth hormone (hGH, or somatotropin) in transfected Nicotiana benthamiana leaves. The system is based on a recently described plant virus-based modular expression vector [Gleba, Y., Marillonnet, S. and Klimyuk, V. (2004) Engineering viral expression vectors for plants: the 'full virus' and the 'deconstructed virus' strategies. Curr. Opin. Plant Biol. 7, 182-188; Marillonnet, S., Giritch, A., Gils, M., Kandzia, R., Klimyuk, V. and Gleba, Y. (2004) In planta engineering of viral RNA replicons: efficient assembly by recombination of DNA modules delivered by Agrobacterium. Proc. Natl. Acad. Sci. USA, 101, 6852-6857], and represents a simple and fast alternative to stable transformation. By using various combinations of provector modules, hGH was produced in three compartments of the cell: the apoplast, the chloroplast and the cytosol. We found that targeting to the apoplast provided the highest amount of correctly processed and biologically active hGH, with a yield of up to 10% of total soluble protein or 1 mg per gram of fresh weight leaf biomass. These results indicate that the use of viral vectors for high-yield production of human therapeutic proteins in plants by transient expression provides an attractive alternative to production protocols using standard expression vectors in transgenic or transplastomic plants.

Producing Human Therapeutic Proteins in Plastids

Plastid transformation technology is set to become a major player in the production of human therapeutic proteins. Protein expression levels that can be achieved in plant plastids are hundreds of times greater than the expression levels generally obtained via nuclear transformation. Plastids can produce human proteins that are properly folded and are biologically active. Effective protein purification strategies and strategies that can achieve inducible plastid gene expression are being developed within the system. Plastid transformation technology has been extended to edible plant species, which could minimize down-stream processing costs and raises the possibility of "edible protein therapies". The system is limited by the fact that plastid-produced proteins are not glycosylated and that, at the moment, it can be difficult to predict protein stability within the plastid. The high level of protein expression that can be obtained in plastids could make it possible to produce high-value therapeutic proteins in plants on a scale that could be accommodated in contained glasshouse facilities and still be economically viable. Growing plastid-transformed plants under contained conditions, and coupled with the level of bio-safety conferred by maternal inheritance of plastid transgenes, would address many of the social and environmental concerns relating to plant based production of human therapeutic proteins.

Modification of Plant N‐Glycans Processing: The Future of Producing Therapeutic Protein by Transgenic Plants

AbstractFor Abstract see ChemInform Abstract in Full Text.

Modification of plant N-glycans processing: The future of producing therapeutic protein by transgenic plants

Transgenic plants are regarded as one of the most promising systems for the production of human therapeutic proteins. The number of therapeutic proteins successfully produced in plants is steadily arising. However, the glycoproteins normally produced from plants are not the same as native therapeutic proteins produced from mammals or humans. In addition to in vitro enzymatic modeling glycoproteins, there are two gene manipulation strategies to humanize plant N-glycans connected to the glycoproteins. One is retaining the recombinant glycoproteins in endoplasmic reticulum (ER), the site where few specific modifications of N-glycans occurs. The other is inhibiting the plant endogenous Golgi glycosyltransferase and/or adding new glycosyltransferase from mammalians. In this review, the biosynthesis of N-glycans in plants, the modification of the plant N-glycans processing will be discussed.

Correction: Corrigendum: Advances in the production of human therapeutic proteins in yeasts and filamentous fungi

Nat. Biotechnol. 22, 1409–1414 (2004) The figure below replaces Figure 3 on page 1413. The new figure corrects errors in the structures and the key of the old figure.

Advances in the production of human therapeutic proteins in yeasts and filamentous fungi

Yeast and fungal protein expression systems are used for the production of many industrially relevant enzymes, and are widely used by the research community to produce proteins that cannot be actively expressed in Escherichia coli or require glycosylation for proper folding and biological activity. However, for the production of therapeutic glycoproteins intended for use in humans, yeasts have been less useful because of their inability to modify proteins with human glycosylation structures. Yeast glycosylation is of the high-mannose type, which confers a short in vivo half-life to the protein and may render it less efficacious or even immunogenic. Several ways of humanizing yeast-derived glycoproteins have been tried, including enzymatically modifying proteins in vitro and modulating host glycosylation pathways in vivo. Recent advances in the glycoengineering of yeasts and the expression of therapeutic glycoproteins in humanized yeasts have shown significant promise, and are challenging the current dominance of therapeutic protein production based on mammalian cell culture.

Enzyme replacement therapy in late-onset Pompe's disease: a three-year follow-up.

Pompe's disease is an autosomal recessive myopathy. The characteristic lysosomal storage of glycogen is caused by acid alpha-glucosidase deficiency. Patients with late-onset Pompe's disease present with progressive muscle weakness also affecting pulmonary function. In search of a treatment, we investigated the feasibility of enzyme replacement therapy with recombinant human alpha-glucosidase from rabbit milk. Three patients (aged 11, 16, and 32 years) were enrolled in the study. They were all wheelchair-bound and two of them were ventilator dependent with a history of deteriorating pulmonary function. After 3 years of treatment with weekly infusions of alpha-glucosidase, the patients had stabilized pulmonary function and reported less fatigue. The youngest and least affected patient showed an impressive improvement of skeletal muscle strength and function. After 72 weeks of treatment, he could walk without support and finally abandoned his wheelchair. Our findings demonstrate that recombinant human alpha-glucosidase from rabbit milk has a therapeutic effect in late-onset Pompe's disease. There is good reason to continue the development of enzyme replacement therapy for Pompe's disease and to explore further the production of human therapeutic proteins in the milk of mammals.

GENENCOR SLATES BIOPHARMA PLANT

ENZYMES PRODUCER GENencor International is the latest company to see opportunity in a projected shortfall in biopharmaceutical manufacturing capacity The firm, which is developing a health care portfolio to complement its traditional industrial enzymes business, intends to build a 22,000-sq-ft facility for the production of human therapeutic proteins. Debby Jo Blank, chiefbusiness officer for health care, says Genencor expects to be making clinical-trial quantities of products in its pipeline within 24 months. The first is a hepatitis vaccine, in development with .the five year-old vaccine firm Epimmune, that would be produced via microbial expression—the method used to make enzymes. The second product is expected to be one of several monoclonal antibodies in development with Seattle Genetics. It would be produced in mammalian cell culture. Genencor also intends to produce for third parties in need of capacity We hope to be able to form alliances with a small number of companies where we bring manufactur...

High-yield production of a human therapeutic protein in tobacco chloroplasts.

Transgenic plants have become attractive systems for production of human therapeutic proteins because of the reduced risk of mammalian viral contaminants, the ability to do large scale-up at low cost, and the low maintenance requirements. Here we report a feasibility study for production of a human therapeutic protein through transplastomic transformation technology, which has the additional advantage of increased biological containment by apparent elimination of the transmission of transgenes through pollen. We show that chloroplasts can express a secretory protein, human somatotropin, in a soluble, biologically active, disulfide-bonded form. High concentrations of recombinant protein accumulation are observed (>7% total soluble protein), more than 300-fold higher than a similar gene expressed using a nuclear transgenic approach. The plastid-expressed somatotropin is nearly devoid of complex post-translational modifications, effectively increasing the amount of usable recombinant protein. We also describe approaches to obtain a somatotropin with a non-methionine N terminus, similar to the native human protein. The results indicate that chloroplasts are a highly efficient vehicle for the potential production of pharmaceutical proteins in plants.