**3. Optimization of plant expression levels**

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

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

**59**

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

tissue-specific promoters are also being used to drive expression of the transgenes to a specific tissue or organ such as the tuber, the seed, and the fruit. Additionally, inducible promoters, whose activities are regulated by either chemical or external stimulus, may equally be used to prevent the lethality problem. Furthermore, transcription factors can also be used as boosters for the promoters to further enhance

Protein stability can be increased by targeting proteins to cell compartments that

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

reduce degradation. Protein targeting also affects the glycan structures added to proteins and the type of extraction and purification steps required to isolate the protein from the plant matrix. Proteins can be targeted to the secretory pathway by an N-terminal signal peptide, which is cleaved off for the release of the protein into the endoplasmic reticulum (ER). Proteins that do not require posttranslational modification, e.g., glycosylation, for their activity, can be targeted to the chloroplast using N-terminal transit peptides [93]. In addition, the target gene can be used to transform chloroplast directly, with highly enhanced protein accumulation. Moreover, posttranslational modifications of the ER lumen can also be avoided by expressing the protein as translational fusion with oleosin protein, which target the expression of the foreign protein to oil bodies of the seeds [28]. Other subcellular compartments like the protein-storing vacuoles are now being explored for recombinant protein

accumulation, as it has been observed in rice seed endosperm [94].

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

the expression level of the transgenes [53].

**4. Downstream processing**

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

tissue-specific promoters are also being used to drive expression of the transgenes to a specific tissue or organ such as the tuber, the seed, and the fruit. Additionally, inducible promoters, whose activities are regulated by either chemical or external stimulus, may equally be used to prevent the lethality problem. Furthermore, transcription factors can also be used as boosters for the promoters to further enhance the expression level of the transgenes [53].

Protein stability can be increased by targeting proteins to cell compartments that reduce degradation. Protein targeting also affects the glycan structures added to proteins and the type of extraction and purification steps required to isolate the protein from the plant matrix. Proteins can be targeted to the secretory pathway by an N-terminal signal peptide, which is cleaved off for the release of the protein into the endoplasmic reticulum (ER). Proteins that do not require posttranslational modification, e.g., glycosylation, for their activity, can be targeted to the chloroplast using N-terminal transit peptides [93]. In addition, the target gene can be used to transform chloroplast directly, with highly enhanced protein accumulation. Moreover, posttranslational modifications of the ER lumen can also be avoided by expressing the protein as translational fusion with oleosin protein, which target the expression of the foreign protein to oil bodies of the seeds [28]. Other subcellular compartments like the protein-storing vacuoles are now being explored for recombinant protein accumulation, as it has been observed in rice seed endosperm [94].
