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Recombinant proteins continue to be a focus of research due to their importance in the biotechnology and biopharmaceutical industries. Since the approval of insulin in 1982 as the first recombinant biopharmaceutical protein produced in Escherichia coli (E. coli), the latter has maintained an annual growth rate of 10-15%. This continuous growth is possible because bottlenecks in the manufacturing process are constantly being overcome. For example, the emergence of monoclonal antibodies (mAbs) as a new product class in 1986 led to the development of Chinese hamster ovary (CHO) cells as one of the dominant expression systems in the biopharmaceutical industry. However, antibody-drug conjugates (ADCs) are antibodies covalently linked to highly toxic small molecule drugs for cancer treatment, which cannot be synthesized by human cells. Therefore, nucleotide-based drugs are also not suitable for such applications. Just as CHO cells have replaced bacteria for the production of new mAbs, plants and plant cell cultures (PCCs) are more suitable for the production of toxic products, reflecting the additional compartmentalization of plant cells, different signaling pathways, and the lack of targets for many proteins that are toxic in mammals. Safety, speed, and sustainability are additional benefits of plant cells. Importantly, biopharmaceutical proteins can be produced in plants and PCCs in compliance with good manufacturing practices (GMP), as demonstrated for mAbs, vaccines, and enzyme replacement therapies.
Plants can express proteins that are toxic when expressed in mammalian or bacterial cells. This is because the molecular target of the toxic protein may not be present in plants, and even if it is present, the toxic protein can be sequestered in subcellular compartments such as the vacuole, which separates the product from its target. For example, ribosome inactivating proteins (RIPs) kill mammalian cells but can be safely produced in plants. This was demonstrated for the lectin viscumin, which is produced in Nicotiana benthamiana in a purified yield of approximately 7 mg kg^-1 fresh weight (FM). The plant-derived protein was approximately three times more active than that expressed in the same bacteria, probably because the latter does not add the sugar structures necessary for the efficient internalization and intracellular trafficking of the second class of RIPs.
Figure 1. Various plant-based expression platforms for the production of recombinant proteins.
RIPs have also been successfully produced in plants fused to antibodies as drug candidates for cancer therapy. The first RIT produced in plant cells was a single-chain fragment variable (scFv) antibody fused to the type I RIP bryodin 1 from Bryonia dioica. Further examples include a truncated version of Pseudomonas exotoxin A expressed in Lactuca sativa and a scFv-RC-RNase fusion protein produced in transgenic tobacco (Nicotiana tabacum) at a yield of approximately 2 mg kg^-1 FM, which is intended to treat hepatocellular carcinoma.
Another promising product category for plant molecular agriculture is derived from plant viruses and includes plant virus-like particles (VLPs) that do not contain nucleic acids, and plant virus nanoparticles (VNPs) that retain nucleic acids. These products are based on plant virus capsids that spontaneously assemble from multiple identical or different coat protein subunits. Because plant viruses have evolved to replicate and assemble in plants, heterologous VLPs and VNPs assemble efficiently in tobacco cells. VLPs and VNPs can be used as imaging agents, immunomodulators, vaccines, and anticancer therapeutics.
Another complex protein that can be produced in plants is spider silk, which is formed when one or more polypeptides called spidroins assemble into a helical structure. These products are valuable in nanotechnology and biomedicine because of their extraordinary toughness and elasticity. Spidroins can be used for nanoparticle-based drug delivery and for the preparation of hydrogels or 3D tissue implants for wound healing and tissue regeneration. These applications have been validated in animal models. Recombinant spidroins can be produced in transgenic plants such as rice (Oryza sativa) and alfalfa (Medicago sativa), which are more suitable for industrial-scale production than E. coli or mammalian cells.
Plants also offer unique manufacturing advantages for therapeutic glycoproteins. One of the most common post-translational modifications (PTMs) in eukaryotes is N-glycosylation, but this is uncommon in bacteria. Glycosylations produced in eukaryotes and bacteria are structurally different, and achieving a humanized glycosylation profile in bacteria is challenging. In contrast, the glycosylation profile of proteins produced in CHO cells has both N- and O-glycosylations that are similar to human glycosylations. As a result, CHO cells have been favored for the manufacture of glycoproteins, currently accounting for more than 60% of approved biopharmaceutical proteins. Even so, the non-human glycosyl structures formed in CHO cells sometimes have more relevant effects on human manufacturing and cannot always be fine-tuned by changing cell culture conditions. For example, up to 80% of human erythropoietin (EPO) produced by CHO cells was discarded because the non-human glycosylation pattern interfered with its biological activity in the treatment of anemia.
Plant glycosylations are also different from those on human glycoproteins, but this can be advantageous. For example, recombinant human β-glucocerebrosidase, used to treat Gaucher disease (a lysosomal storage disorder), is active when expressed in plants and directed to accumulate in the vacuole. This adds mannose tips to the glycosyl groups, which are recognized by receptors on human macrophages. In contrast, CHO cells extend the glycosyl groups and render the protein inactive unless the glycosyl groups are trimmed in vitro. Similarly, some plant-derived antibodies have been shown to be less immunogenic and more potent than the same proteins expressed in CHO cells.
When the glycosyl structure is critical for the activity of the recombinant protein, the production host can be genetically engineered to favor the production of an appropriate glycosylation profile. This can include overexpression or inhibition of individual enzymes such as sialyltransferases or sialidases. This precision strategy has been used to improve the glycosylation profile of EPO produced in plants. Genetic engineering can even achieve full humanization of plant-derived glycoproteins, which may support the approval of biopharmaceutical proteins where glycosyl groups strongly affect pharmacokinetics or immunogenicity. Plants do not typically add terminal sialic acid to the N-acetylglucosamine (GlcNAc) residue in N-glycosylation, a modification commonly found in humans. However, they do add core α-1,3-fucose and β-1,2-xylose, which are not found in mammals, so several modifications are required to "humanize" the plant glycosylation machinery. These modifications have been achieved through strategies such as overexpression of missing enzymes and the use of tools such as RNA interference (RNAi) and CRISPR/Cas9 to inhibit unwanted reactions.
Although production hosts such as tobacco plants do not compete with food or feed crops, they contain toxic metabolites such as nicotine base, which must be removed in downstream processing (DSP) to comply with GMP regulations. By abolishing nicotine synthesis using CRISPR/Cas9 technology, recombinant protein can be purified without the need for additional steps to remove this metabolite. By selectively knocking out six tobacco genes encoding berberine bridge enzyme-like (BBL) proteins, the final oxidation step in the nicotine synthesis pathway was blocked, resulting in a greater than 99% reduction in nicotine levels.
Further modifications of the host plant can increase product yields and include modifications of the tRNA repertoire, suppression of gene silencing or protein degradation, and optimization of stress tolerance. Other options include reducing endogenous oxidase and protease activities, or co-expressing molecular chaperones to improve protein folding.
Although plant-based expression platforms can produce complex biopharmaceutical proteins, the competitiveness of plant cells compared to traditional microbial and mammalian systems is currently limited by the following challenges: (1) upstream production (USP) yields, (2) DSP recovery, and (3) technological differences from traditional systems designed for microbial and mammalian cells.
A major challenge is the relatively low yields of PCCs. Unlike whole plants, PCCs generally do not require complex clarification processes. This is because the product can be secreted into the culture medium and recovered from it, similar to perfusion processes using mammalian cells, such as those based on tangential flow filtration (TFF) or alternating tangential flow filtration (ATF). Chromatographic purification can be challenging if an affinity-based separation step is not available, which is often the case for non-antibody products. Finally, industry acceptance and willingness to invest in new plant-based expression systems is limited. Barriers to widespread market penetration may include low productivity, high DSP costs, and regulatory barriers.
Creative Biogene has established various plant-based protein expression systems to provide recombinant protein production services for global clients. Using our state-of-the-art platform, we offer a complete service that begins from a specific gene to the purified protein, as well as different options for protein expression systems.
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