**2.3 Transient expression platforms**

Transient expression is a phenomenon that occurs when genes are introduced into plant tissues and are expressed for a short period without stable DNA integration into the genome [3]. Traditionally, transient expression was used to verify expression construct activity and to test recombinant protein stability. This strategy allowed the identification and elimination of initial transformation problems, and thus the prospect of regenerating the desired transgenic lines was significantly improved. Recently, there has been an emergence of transient expression for the commercial production of recombinant pharmaceutical proteins. The advantages of transient expression platforms include the ease of manipulation, speed, low cost, and high yield of proteins. In comparison to transgenic plants, transient expression permits to achieve higher recombinant protein yields because there are no position effects (suppression of transgene expression by the surrounding genomic DNA following integration) [70].

Transient expression systems utilize the beneficial properties of plant pathogens to infect plants, spread systemically, and express transgenes at high levels, causing the rapid accumulation of recombinant proteins [8]. Currently, the major transient expression platforms are based on *Agrobacterium tumefaciens*, plant viruses, or hybrid vectors that utilize components of both (magnICON® technology).

The agroinfiltration method involves the vacuum infiltration of a suspension of recombinant *A. tumefaciens* into the plant leaf tissue, with the transgenes being then expressed from the uninterrupted T-DNA [8, 71]. Using this method, milligram amounts of recombinant protein are produced within a few weeks without the need to select transgenic plants, a process that takes months to years to be completed. This system has been commercially developed in tobacco [72] and alfalfa [73] but is also applicable to other crops such as lettuce [74], potato [75], and *Arabidopsis* [76]. An advantage of *Agrobacterium*-mediated transient expression is the fact that it allows to produce in plants complex proteins assembled from subunits [70].

Another transient expression technology is based on the use of plant viruses. In this technology, the gene of interest is inserted among viral replicating elements, episomically amplified and subsequently translated in the plant cell cytosol [77]. To date, the most efficient and high-yielding platforms have been developed using RNA viruses [78]. These plant viruses include *Tobacco mosaic virus* (TMV), *potato virus X* (PVX), and *Cowpea mosaic virus* (CPMV) (reviewed in [8]). The advantages of virus-based production include the rapid recombinant protein expression, the systemic spread of the virus, and the fact that multimeric proteins such as antibodies can also be produced by coinfecting plants with noncompeting vectors derived from different viruses [79, 80]. Transient expression vectors based on virus have been used to express peptides and long polypeptides (at least 140 amino acids long) as fusions to the coat protein, resulting in the assembly of chimeric virus particles (CVPs) displaying multiple copies of the peptide or polypeptide on its surface [77, 81].

**57**

therapies.

*Prospects for the Production of Recombinant Therapeutic Proteins and Peptides in Plants…*

Transient expression based in plant viruses has been commercially adopted by the now-closed Large Scale Biology Corporation (Vacaville, USA) that used a TMVbased vector for the production of patient-specific idiotype vaccines for the treatment of B-cell non-Hodgkin's lymphoma, which had successfully passed the phase I

Finally, the third transient expression strategy is based on hybrid systems that incorporate components of the T-DNA transfer and virus replication systems [3]. These hybrid systems use deconstructed viruses obtained by removing the coat protein (responsible for systemic movement) of the noncompeting virus strains and use *Agrobacterium* as the vehicle for the systemic delivery of the resulting viral vectors to the entire plant. These systems effectively address most of the major shortcomings of earlier plant-based technologies by providing the overall best combination of the following features: high expression level, high relative yield, low up- and downstream costs, very fast and low-cost R&D, and low biosafety concerns [83]. Consequently, there has been a commercial development based on several hybrid systems. One of most notable examples is the magnICON® system developed by Icon Genetics (https://www.icongenetics.com/) (formerly owned by Bayer Innovation, Dusseldorf, Germany; now a subsidiary of Nomad Bioscience, Halle, Germany), which features a deconstructed *Tobacco mosaic virus* (TMV) genome and *A. tumefaciens* as a delivery vehicle [83]. Another example is the iBioLaunch platform developed by the Fraunhofer Center for Molecular Biotechnology, which also features a deconstructed TMV genome [3]. Finally, the CPMV-HT platform is based on a deleted version of *Cowpea mosaic virus* RNA-2 and also allows the "hypertrans-

Examples of therapeutic recombinant proteins produced in these platforms have been generally reviewed in [3]. Recombinant protein production using transient expression is now being mobilized to a large scale with several companies developing scalable, automated plant-based GMP biomanufacturing facilities to efficiently produce large amounts of pharmaceuticals within weeks. Such facilities include the ones of the Fraunhofer Center for Molecular Biotechnology (Newark, DE) (https:// www.fraunhofer.org/), Medicago Inc. (Quebec, Canada) (http://www.medicago. com/), Icon Genetics (Bayer; Halle, Germany) (http://www.icongenetics.com/), Texas A & M (College Station, TX), and Kentucky BioProcessing LLC (Owensboro,

In conclusion, the ability of transient plant expression systems to produce large

quantities of recombinant protein, coupled to the use of current technology to increase yields, and the many promising technical solutions seems to be favorable compared with mammalian- or insect cell-based systems in quality, cost, and scale [19]. In case of emerging threats, transient platforms are advantageous since they produce large amounts of recombinant proteins rapidly (milligram quantities per plant within a few days) and can be scaled up quickly, currently providing the only reliable platform for rapid response situations [9]. During the H1N1 pandemic, the first batches of H1N1 virus-like particles (VLPs) could be produced by Medicago Inc. as soon as 3 weeks after the Centers for Disease Control and Prevention released the new influenza hemagglutinin sequence [73]. Similar lead times were reported for the H5N1 VLP vaccine [84]. Recently, the application of tobacco plantbased transient production systems, at Kentucky BioProcessing (KBP), to produce antibody lots against Ebola, was shown to significantly decrease the amount of time required for production over traditional methods, increase the quantity of antibody produced, and reduce the cost of manufacturing. Finally, at the other end of the market scale, transient expression platforms are economical for the production of pharmaceuticals for very small markets, such as orphan diseases and individualized

lation" of recombinant proteins without virus spreading [8].

KY) (http://www.kbpllc.com/) [19].

*DOI: http://dx.doi.org/10.5772/intechopen.84419*

clinical trials [82].

## *Prospects for the Production of Recombinant Therapeutic Proteins and Peptides in Plants… DOI: http://dx.doi.org/10.5772/intechopen.84419*

Transient expression based in plant viruses has been commercially adopted by the now-closed Large Scale Biology Corporation (Vacaville, USA) that used a TMVbased vector for the production of patient-specific idiotype vaccines for the treatment of B-cell non-Hodgkin's lymphoma, which had successfully passed the phase I clinical trials [82].

Finally, the third transient expression strategy is based on hybrid systems that incorporate components of the T-DNA transfer and virus replication systems [3]. These hybrid systems use deconstructed viruses obtained by removing the coat protein (responsible for systemic movement) of the noncompeting virus strains and use *Agrobacterium* as the vehicle for the systemic delivery of the resulting viral vectors to the entire plant. These systems effectively address most of the major shortcomings of earlier plant-based technologies by providing the overall best combination of the following features: high expression level, high relative yield, low up- and downstream costs, very fast and low-cost R&D, and low biosafety concerns [83]. Consequently, there has been a commercial development based on several hybrid systems. One of most notable examples is the magnICON® system developed by Icon Genetics (https://www.icongenetics.com/) (formerly owned by Bayer Innovation, Dusseldorf, Germany; now a subsidiary of Nomad Bioscience, Halle, Germany), which features a deconstructed *Tobacco mosaic virus* (TMV) genome and *A. tumefaciens* as a delivery vehicle [83]. Another example is the iBioLaunch platform developed by the Fraunhofer Center for Molecular Biotechnology, which also features a deconstructed TMV genome [3]. Finally, the CPMV-HT platform is based on a deleted version of *Cowpea mosaic virus* RNA-2 and also allows the "hypertranslation" of recombinant proteins without virus spreading [8].

Examples of therapeutic recombinant proteins produced in these platforms have been generally reviewed in [3]. Recombinant protein production using transient expression is now being mobilized to a large scale with several companies developing scalable, automated plant-based GMP biomanufacturing facilities to efficiently produce large amounts of pharmaceuticals within weeks. Such facilities include the ones of the Fraunhofer Center for Molecular Biotechnology (Newark, DE) (https:// www.fraunhofer.org/), Medicago Inc. (Quebec, Canada) (http://www.medicago. com/), Icon Genetics (Bayer; Halle, Germany) (http://www.icongenetics.com/), Texas A & M (College Station, TX), and Kentucky BioProcessing LLC (Owensboro, KY) (http://www.kbpllc.com/) [19].

In conclusion, the ability of transient plant expression systems to produce large quantities of recombinant protein, coupled to the use of current technology to increase yields, and the many promising technical solutions seems to be favorable compared with mammalian- or insect cell-based systems in quality, cost, and scale [19]. In case of emerging threats, transient platforms are advantageous since they produce large amounts of recombinant proteins rapidly (milligram quantities per plant within a few days) and can be scaled up quickly, currently providing the only reliable platform for rapid response situations [9]. During the H1N1 pandemic, the first batches of H1N1 virus-like particles (VLPs) could be produced by Medicago Inc. as soon as 3 weeks after the Centers for Disease Control and Prevention released the new influenza hemagglutinin sequence [73]. Similar lead times were reported for the H5N1 VLP vaccine [84]. Recently, the application of tobacco plantbased transient production systems, at Kentucky BioProcessing (KBP), to produce antibody lots against Ebola, was shown to significantly decrease the amount of time required for production over traditional methods, increase the quantity of antibody produced, and reduce the cost of manufacturing. Finally, at the other end of the market scale, transient expression platforms are economical for the production of pharmaceuticals for very small markets, such as orphan diseases and individualized therapies.

*Genetic Engineering - A Glimpse of Techniques and Applications*

**2.3 Transient expression platforms**

following integration) [70].

[59] demonstrated the capacity of chloroplasts to fold and assemble oligomeric proteins correctly. Other therapeutic proteins expressed in tobacco chloroplasts include interferons alpha-2a and alpha-2b [60, 61] and anti-cancer therapeutic agents such as human soluble tumor necrosis factor (TNF) [62] and azurin [63]. Recently, chloroplast transformation of lettuce has also been developed [64, 65] to provide oral delivery transplastomic systems [66, 67]. Several therapeutic proteins were produced in lettuce chloroplast, namely, proinsulin [66, 67], tuberculosis vaccine antigens [68], and human thioredoxin 1 protein [69]. The chloroplast production platform has yet to achieve commercial success, though the referred developments in this field

augur a promising future for therapeutic protein production in chloroplasts.

Transient expression is a phenomenon that occurs when genes are introduced into plant tissues and are expressed for a short period without stable DNA integration into the genome [3]. Traditionally, transient expression was used to verify expression construct activity and to test recombinant protein stability. This strategy allowed the identification and elimination of initial transformation problems, and thus the prospect of regenerating the desired transgenic lines was significantly improved. Recently, there has been an emergence of transient expression for the commercial production of recombinant pharmaceutical proteins. The advantages of transient expression platforms include the ease of manipulation, speed, low cost, and high yield of proteins. In comparison to transgenic plants, transient expression permits to achieve higher recombinant protein yields because there are no position effects (suppression of transgene expression by the surrounding genomic DNA

Transient expression systems utilize the beneficial properties of plant pathogens to infect plants, spread systemically, and express transgenes at high levels, causing the rapid accumulation of recombinant proteins [8]. Currently, the major transient expression platforms are based on *Agrobacterium tumefaciens*, plant viruses, or hybrid vectors that utilize components of both (magnICON® technology).

The agroinfiltration method involves the vacuum infiltration of a suspension of recombinant *A. tumefaciens* into the plant leaf tissue, with the transgenes being then expressed from the uninterrupted T-DNA [8, 71]. Using this method, milligram amounts of recombinant protein are produced within a few weeks without the need to select transgenic plants, a process that takes months to years to be completed. This system has been commercially developed in tobacco [72] and alfalfa [73] but is also applicable to other crops such as lettuce [74], potato [75], and *Arabidopsis* [76]. An advantage of *Agrobacterium*-mediated transient expression is the fact that it allows to produce in plants complex proteins assembled from subunits [70].

Another transient expression technology is based on the use of plant viruses. In this technology, the gene of interest is inserted among viral replicating elements, episomically amplified and subsequently translated in the plant cell cytosol [77]. To date, the most efficient and high-yielding platforms have been developed using RNA viruses [78]. These plant viruses include *Tobacco mosaic virus* (TMV), *potato virus X* (PVX), and *Cowpea mosaic virus* (CPMV) (reviewed in [8]). The advantages of virus-based production include the rapid recombinant protein expression, the systemic spread of the virus, and the fact that multimeric proteins such as antibodies can also be produced by coinfecting plants with noncompeting vectors derived from different viruses [79, 80]. Transient expression vectors based on virus have been used to express peptides and long polypeptides (at least 140 amino acids long) as fusions to the coat protein, resulting in the assembly of chimeric virus particles (CVPs) displaying multiple copies of the peptide or polypeptide on its surface [77, 81].

**56**
