Harnessing Molecular Farming’s Potential 

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What Is Molecular Farming?

Molecular farming is a technique that uses plants as factories to produce recombinant protein products such as antibodies and vaccines for pharmaceutical purposes.¹ “Molecular farming is the art and science of making proteins of pharmaceutical interest in plants, as an alternative production system to the more conventional mammalian cells, yeast, and bacteria,” explained Edward Rybicki, a biotechnologist and former director of the University of Cape Town’s Biopharming Research Unit.

Advantages of molecular farming over conventional systems

In the 1980s, scientists synthesized the first recombinant human insulin in the bacterium Escherichia coli.² Over the years since, researchers have explored many alternative expression systems to synthesize similar value-added biological compounds, such as insects, bacterial species other than E. coli, fungi, yeast, plant cells in culture, and transgenic plants and animals.³

Although conventional bacteria have several strengths, including high biological compound yield and inexpensive expression systems, they lackeukaryotic chaperones that are essential for the proper functioning of recombinant animal proteins.⁴ Additionally, the inability of bacterial systems to carry out relevant posttranslational modifications such as glycosylation leads to inappropriate protein folding.⁵ Although protein modification occurs in yeast, the glycosylation pattern differs from mammalian cells. Unlike prokaryotic and yeast platforms, plants perform the same posttranslational modifications as mammalian cells and express complex proteins such as monoclonal antibodies (mAbs). “Plants are, strangely enough, more closely related to humans than yeast are,” said Rybicki.

Finally, although transgenic animals and mammalian cell culture systems promote optimal expression due to correct protein modifications, they are expensive, which hinders efficient large-scale production. In comparison to conventional multicellular platforms, plants offer superior speed, higher safety, and scalability for biopharmaceutical production.

Molecular Farming Techniques 

“Molecular farming started out with transgenic plants,” said Rybicki. Scientists developed several strategies to produce transgenic plants with either permanent (stable) expression systems, or temporary (transient) expression systems.⁶ These methods have their own strengths and limitations, for instance, the stable gene expression method is useful for large-scale production of recombinant proteins, but it is more time consuming than transient expression systems, requiring months to establish.

Stable genetic transformation

Researchers incorporate recombinant genes into plant cell genomes to produce stable proteins using nuclear or chloroplast transformation methods, which incorporate the gene of interest (GOI) into the nuclear chromosome or chloroplast DNA, respectively.⁷

The nuclear transformation technique enables a random insertion of genes into the plant nucleus via inoculation for Agrobacterium-mediated transformation, polyethylene glycol for protoplast-mediated transformation, or high velocity gold or tungsten-coated particles for biolistic transformation methods.^8-10 Two key advantages of nuclear transformation strategies are multiple recombinant protein synthesis and large-scale production.⁷ However, this technique includes the risk of endogenous gene silencing and unstable recombinant protein expression due to sub-optimal gene insertion positioning. 

Using similar methods, researchers may also insert a GOI into the chloroplast chromosome to express the recombinant protein specifically in leaf tissue.⁷ This process enables greater recombinant protein yield due to increased GOI expression in the chloroplast genome, which is abundant in leaf tissues. 

Transient gene expression

Transient gene expression has revolutionized the field of molecular farming.⁸ Researchers use this method to produce target protein more rapidly than the stable genetic transformation methods. They commonly use an Agrobacterium-mediated infiltration method for transient gene expression. 

Agrobacterium-mediated infiltration uses the soil microorganism Agrobacterium tumefaciens to transfer the DNA into the plants. In this technique, researchers introduce Agrobacterium culture in leaves using a needleless syringe or vacuum, and harvest these leaves within 3 to 4 days post infiltration to extract proteins. “All you have to do is get enough bacteria into plant leaves to get DNA transferred, so that nearly every cell in the plant is making what you want without being transgenic,” explained Rybicki. 

Because researchers deliver transgenes episomally to the nucleus of the cell such that it does not integrate with host’s chromosome, transient gene expression methods have no positional effects, unlike the nuclear transformation method. Another advantage of this technique is rapid recombinant protein expression within 18 to 48 hours, which remains steady for approximately 10 days.¹¹

Expression in suspension cell cultures

Scientists develop plant suspension cultures by growing individual cells from callus, a rapidly proliferating mass of unorganized parenchyma cells.⁸ Subsequently, they co-culture the cells with A. tumefaciens to produce recombinant proteins. Researchers use tobacco, Arabidopsis thaliana, alfalfa, tomato, and carrot cells to produce recombinant proteins and other valuable metabolites. This technique is advantageous because plant cells grow faster in suspension, which enables large-scale production of recombinant proteins.

Molecular Farming for Biopharmaceutical Production

Scientists produce vaccines, antibodies, and therapeutics through plant molecular farming.⁸

Vaccines

Vaccines activate the immune system and confer protection against harmful pathogens. Scientists have created vaccines from virus-like particles (VLP), which are non-pathogenic nanoscale structures containing viral antigens, similar to a viral capsid that triggers an immunogenic reaction.¹² 

Additionally, transient expression systems in Nicotiana benthamiana enable plant-based vaccine production, including influenza vaccines that have reached clinical trials.¹³ After successful plant-based vaccine development against the Newcastle virus infection in poultry, research interest has considerably increased.¹⁴ Over the years, scientists have created multiple plant-produced vaccines against the hepatitis B virus, influenza, porcine circovirus, and poliovirus.¹⁵

Antibodies

In 1989, scientists first produced immunoglobulin chains in tobacco.¹⁶ Ever since, multiple plant-made antibody therapeutics have been developed such as ZMapp, a plant-produced monoclonal antibody to treat Ebola virus infection.¹⁷

Therapeutic proteins

A key therapeutic breakthrough in plant molecular farming was the successful development of a Gaucher disease treatment. Gaucher disease is a rare genetic disorder associated with mitochondrial enzyme deficit. In 2012, the US Food and Drug Association (FDA) approved a plant-derived recombinant human β-glucocerebrosidase to treat Gaucher’s disease.¹⁸ Researchers synthesized this therapeutic recombinant enzyme in a stably transformed carrot cell suspension culture. 

Scientists have also focused on producing cytokines in plant systems because of their multiple functions associated with cell proliferation, differentiation, and migration.⁸ For example, the coat protein (CP)-deficient bamboo mosaic virus (BaMV) vector enabled a cost-efficient high expression of human interferon-γ levels, an important cytokine-based therapeutic protein, in N. benthamiana.¹⁹ Furthermore, scientists have produced blood coagulation FVIII factor in the chloroplasts of lettuce and N. tabacum, which could be tremendously beneficial to treat hemophilia, a bleeding disorder.²⁰

Molecular Farming Advantages and Challenges in Producing Biopharmaceuticals

In comparison to conventional biomanufacturing methods, molecular farming stands out for its numerous advantages including the following.²¹

• Cost-effectiveness: Molecular farming can significantly reduce production costs as it requires limited facilities. In comparison to any other eukaryotic system, plant growth is inexpensive, as it only requires water and sunlight.
• Scalability: Molecular farming can easily scale up the production of biopharmaceuticals, particularly due to the advent of transient expression technologies that enable protein expression within a few days of the DNA construct availability. 
• Sustainability: In principle, plants can grow without the extensive use of chemicals and can significantly reduce environmental impacts of biomanufcaturing.²²
• Safety: Molecular farming in plants has a lower risk of contamination, which reduces the risk of batch loss, unlike conventional bacterial systems.

Many barriers inhibit the proper utilization of molecular farming for large-scale biopharmaceutical production. The key challenges involved with biomanufacturing in plants include the following.¹⁹

• Low productivity: In comparison to mammalian and microbial production systems, the protein yield in plant-based systems is relatively low. Scientists address this limitation by improving various internal (e.g., protein expression) and external factors (e.g., nutritional and physical cultivation parameters).
• Regulatory compliance: In comparison to industrial microbial and mammalian cell expression systems, the road to regulatory and legal approval for biological products derived from plant molecular farming has more uncertainties. Industries tend to perceive molecular farming as a risk and prefer to adopt the tested platforms.
• Public perception: Plant molecular farming applicability significantly depends on public perception.²³ Although some people have accepted that genetic modifications in molecular farming are safe, some continue to be skeptical. Transparency of bioengineering techniques could reduce the negative public perception.
• Reproducibility: There are several challenges producing recombinant proteins in high abundance, such as precision, stability, and repeatability of gene expression in plant systems. It is important to optimize the stability of recombinant proteins through a variety of regulatory DNA elements such as promoters, enhancers, and terminators for robust and stable transgene expression.

Future Outlook

Future research should identify novel proteins and metabolites synthesized by plants and further explore using plant-based molecular farming to produce therapeutic proteins. “It’s in that sort of area, making antibodies as therapeutics, that I think that this field is going to boom,” Rybicki said. “If you want to make limited scale therapeutics for orphan diseases or less widely circulating diseases, you’re going to have to use something like plants because it’s just not economical enough for anybody else to do.” The advent of new gene-editing technologies such as clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 also heralds a new era for molecular farming, promising greater efficiency, precision, stability, and reduced cost. 

Many developing countries struggle to provide adequate treatments due to high production costs.²⁴ The persistent efforts in mastering molecular farming techniques enhance the possibility of plants becoming multi-dimensional resources, moving beyond providers of basic sustenance through food and into producing therapeutic proteins and compounds to cure diseases. 

FAQ:

What is molecular farming?

• Molecular farming, also known as molecular pharming or biopharming, is a technique that uses plants or plant suspension cells to express recombinant genes that produce therapeutic proteins and other value-added products.

What is an example of molecular farming?

• The plant-derived recombinant human β-glucocerebrosidase aids in treating Gaucher’s disease type 1.

What is the difference between molecular farming and precision fermentation?

• The key difference between molecular farming and precision fermentation is that the former utilizes living plants while the latter uses microorganisms such as yeast and bacteria for fermentation in a bioreactor. 

What are the products of molecular farming?

• Plant molecular farming enables the production of vaccines, antibodies, and therapeutic proteins.