**4. Downstream processing**

*Genetic Engineering - A Glimpse of Techniques and Applications*

Plant cell suspension cultures grow as individual cells or small aggregates and are usually derived from *callus* tissue by the disaggregation of friable callus pieces in shake bottles and are later scaled up for bioreactor-based production. Recombinant pharmaceutical protein production is achieved using transgenic explants to derive the cultures or by transforming the cells after disaggregation, usually by co-cultivation with *A. tumefaciens*. The co-cultivation of plant cell suspensions and recombinant *A. tumefaciens* has also been used for the transient expression of proteins [85]. Since these plant cell suspension cultures are grown in sterile contained environments, they provide a cGMP-compatible production environment that is more acceptable to the established pharmaceutical industry and regulatory authorities [3, 86]. These systems have added benefits of complex protein processing compared to bacteria and yeasts and increased safety compared to mammalian cell systems, which can harbor human pathogens. Another advantage of plant suspension cultures is the very low maintenance cost in comparison to other fermenter-based eukaryotic systems such as mammalian or insect cells. Moreover, the possible secretion of the target protein into the culture medium simplifies downstream processing and purification procedures [87, 88]. Nevertheless, plant cell cultures also have some limitations such as poor growth rates, somaclonal variation (particularly due to chromosomal rearrangements, common in plant cell cultures generated by *calli*), and gene silencing, together with the inhibition of product formation at high cell densities, formation of aggregates, cell wall growth, as well as shear-sensitivity for some species [89]. However, high levels of functional recombinant protein in plant cell suspension cultures were already obtained [87]. Besides, the previously mentioned first licensed recombinant pharmaceutical protein, Elelyso™, was produced in plant cell suspension cultures (reviewed in [88]). Tobacco has been the most popular source of suspension cells for recombinant protein production. Tobacco plants proliferate rapidly and are easy to transform, but other plant species have also been used to generate suspension cells, including rice and *Arabidopsis thaliana,* alfalfa, soybean, tomato, *Medicago truncatula*, and carrot [85, 88, 90]. Carrot suspension cells have been used by the aforementioned Protalix Biotherapeutics to produce a recombinant glucocerebrosidase. This case of commercial success shows that suspension cell cultures have potential as a viable system for large-scale protein production. Recently, carrot callus cultures, expressing epitopes from the cholesteryl ester transfer protein, were

accessed for the potential of becoming an atherosclerosis oral vaccine [91].

The lower expression levels in comparison to the established biopharmaceutical platforms were one of the major obstacles for the commercialization of molecular pharming [9]. Therefore, numerous techniques have been developed to enhance protein expression, including codon optimization of protein sequences, to match the preferences of the host plant, targeting subcellular compartments that allow proteins to accumulate in a stable form; the use of strong, tissue-specific promoters;

Protein synthesis can be increased by optimizing the components of the expression construct to maximize transcription, mRNA stability, and translation or by diminishing the impact of epigenetic phenomena that inhibit gene expression [92]. In this field, the general strategy is to use strong and constitutive promoters, such as the cauliflower mosaic virus 35S RNA promoter (CaMV 35S) and maize ubiquitin-1 promoter (ubi-1), for dicots and monocots, respectively. However, organ- and

**3. Optimization of plant expression levels**

and the testing of different plant species and systems [25].

**2.4 Callus and plant cell suspension cultures**

**58**

In the early years of molecular pharming, scientific studies were focused on demonstrating that plants could produce adequate quantities of recombinant pharmaceutical proteins and confer an oral delivery means. This led to downstream processing and the costs associated to it being basically overlooked. Downstream processing is now known to be an economically critical part of biomanufacturing processes (it can account for up to 80% of the total cost in a therapeutic protein production line) and also to be a key component of the regulatory process for evaluating the safety of pharmaceutical products [7]. The goal and the general steps for downstream processing are similar between plant and other expression systems: to recover the maximal amount of highly purified target protein with the minimal number of steps and at the lowest cost. The basic steps for downstream processes include tissue harvesting, protein extraction, purification, and formulation [22]. However, since in molecular pharming the costs of downstream processing are product-specific rather than platform-specific, the evaluation of downstream processing strategies and costs associated to it has to be done on a case-by-case basis. Nevertheless, even if unit operations have to be developed based on the properties of the product, others have to be developed based on the properties of the expression host. Plants produce process-related contaminants that require specific processing steps to ensure removal of fibers, oils, superabundant plant proteins such as RuBisCO, and potentially toxic metabolites such as the alkaloid nicotine in tobacco [8]. These secondary metabolites can be recovered from plant cells or tissues using methods such as adsorption, precipitation, and chromatography, often requiring phase partitioning and the use of mixtures of organic solvents. Several approaches have been used to facilitate downstream processing, including secretion of recombinant proteins, eliminating the plant cell disruption step; targeting of proteins into the protein bodies, oil bodies, or plastoglobules; and the use of affinity tags such as poly-histidine tags with the target protein, allowing protein purification by affinity chromatography [25]. In addition, oral delivery of whole plants or crude extracts containing the pharmaceutical relevant proteins can also be a way to simplify downstream processing and to

easily distribute medicines to those in need. Furthermore, the optimization of plant's expression level can also ease downstream processing, with higher protein concentrations conducting to higher protein volumes [7].

Finally, several purification strategies have been investigated to separate target transgenic proteins from host plant proteins, which are tailored for each individual protein based on its solubility, size, pI, charge, hydrophobicity, or affinity to specific ligands, and the parallel characteristics of plant host proteins. Chromatographic methods, such as affinity chromatography, have been the most extensively used. However, recently increasing attention is being paid to nonchromatographic methods to provide alternatives for large-scale production [22].
