**2. Plant platforms for the production of therapeutic proteins and peptides**

The continuous development of genetic engineering technologies for plants has resulted in an expansion of well-established plant-based platforms [18]. Molecular

*Genetic Engineering - A Glimpse of Techniques and Applications*

confirmed native structure in tobacco and potato [6].

transgenic animals. Nowadays, the expression is mainly employed to the production of recombinant pharmaceutically relevant proteins or secondary products in plants [1–3]. The roots of molecular pharming can be traced back to the mid-1980s when plants started to be genetically engineered to act as bioreactors that produced pharmaceutically relevant proteins. Barta et al. [4] demonstrated that tobacco and sunflower callus tissues were capable of expressing transcripts of a human growth hormone fusion gene. Although no protein was detected, this was the first report of plants expressing human genes and established plants as a potential production system for recombinant therapeutic proteins. Later on, the expression of a full-sized IgG in tobacco [5] was a major breakthrough since it revealed the ability of plants to produce complex functional mammalian proteins of pharmaceutical relevance. In 1990, the "authenticity" of plant-derived recombinant proteins was proved even further with the production of the first human protein (serum albumin) with

After several studies that demonstrated the capacity of various plant species and systems to produce recombinant pharmaceutical proteins and peptides, during the 1990s, the field of molecular pharming gained support and interest from the plant biotechnology community. The scientific attention was followed by commercial interest, with many start-up companies being created to capitalize the advantages of plants in relation to the established platforms. These advantages include being a more costeffective, scalable, and safer means of producing pharmaceutically relevant proteins and peptides. In opposition to the fermentation-based traditional platforms that require a massive investment in bioreactors, plant-based production systems can be established with minimal investment and offer a myriad of different hosts and platforms [7]. However, the expectation that plants could easily compete for the market share of some well-established biopharmaceutical platforms, such as Chinese hamster ovary (CHO) cells, and that they could motivate the mainstream pharmaceutical industry to switch platforms was overinflated. The CHO epithelial cell lines are the most commonly used mammalian hosts for industrial production of therapeutic recombinant proteins. The technical limitations of plants, especially their lower yields compared to mammalian cell lines, allied to the colossal existing investment in fermentation infrastructures, the unfavorable public opinion on OGMs, and regulatory uncertainty, lead the mainstream pharmaceutical industry to be cautious and to a consequent stagnation of the molecular pharming field in the 2000s [8, 9]. This situation induced a change of paradigm concerning molecular pharming: the initial vision of a highly scalable and low-cost production system, while still valid, was replaced by the idea of a production system for certain

niche products that are not easily manufactured by conventional systems [8, 9].

Molecular pharming embraces several platforms and technologies with different advantages and limitations, related by their use of plant tissues. Conversely to conventional biopharmaceutical production systems that are based on few selected platforms, particularly the bacterium *Escherichia coli*, yeasts such as *Pichia pastoris*, and mammalian cell lines such as Chinese hamster ovary (CHO) cells [3], pharming platforms range from plant cells or unicellular plants growing in bioreactors to whole plants growing in soil or hydroponic environments. Further, the technologies include stable integration of DNA into the nuclear genome or plastid genome and transient expression by infiltrating leaves with expression vectors based on *Agrobacterium tumefaciens*, plant viruses, or hybrids [3, 8]. This great diversity of molecular pharming confers adaptability and flexibility, allowing the selection of the most suitable platform for each product, but has also conduced to fragmentation. This fragmentation meant that in the early days of molecular pharming there was no driving force to establish molecular pharming as a single competitive platform. Consequently, no actions were made to match the industry requirements for high yields, standardized procedures, and good manufacturing practices (GMP) [7, 9]. More recently, efforts have been made to mimic the mainstream

**50**

pharming encompasses platforms based on stably transformed whole-plants transgene insertion in the nuclear or plastid genome, transient expression using agroinfiltration, viral and hybrid vectors; microalgae and aquatic plants (e.g., duck-weed) stably transformed; and in vitro culture systems (e.g., cell suspensions, hairy roots, and moss protonema) [19]. Each platform has particular advantages and limitations; therefore its selection is done on a case-by-case basis, depending on economic considerations as well as on the product characteristics and intended use [20].
