In the fast-evolving world of biotechnology, protein expression stands as a cornerstone of innovation. The method typically involves manipulating gene expression within an organism to produce large quantities of recombinant proteins. While various host cells can be utilized for expression (bacteria, yeast, fungi, insect cell lines, mammal cell lines, avian cell lines, plant), recent developments have led to an exciting frontier – microalgae.
Powering the Future: Post-translational Modifications in Protein Expression
Post-translational modifications, such as glycosylation, phosphorylation, and disulfide bond formation, play a pivotal role in ensuring the correct assembly and folding of proteins. This is essential for their functionality. Although mammalian systems currently dominate the biomanufacturing industry, several challenges need to be addressed:
- Reducing production costs.
- Accelerating commercialization and manufacturing times.
- Reducing the risks of viral transmission from animal cells to humans.
Emergence of Plant and Algae-Based Platforms
A wave of innovative companies, including PlantForm, iBio, LeafBio, and more, is positioning plants as promising production platforms. Medium- and large-scale cGMP-compliant plant facilities have already begun operations in North America and Europe.
However, the production of biodrugs by transgenic plants faces commercial limitations due to large land requirements, low surface productivity, slow growth cycles, light-dependent production, and the risk of environmental contamination. In light of these challenges, unicellular eukaryotic green algae have emerged as an alternative for producing recombinant proteins, including protein vaccines and therapeutic antibodies.
| Expression system | Example | Advantages | Limitations |
|---|---|---|---|
| Bacteria | E. coli, B. subtilis, B. Calmette-Guerin, P. fluorescens | Production time: short Cost: cost of cultivation medium Scalability: highly scalable but high scale-up costs Product yield: easily produce large amounts of protein, medium yield |
Posttranslational modifications: limited, no glycosylation/ phosphorylation/disulphide bond formation Safety: endotoxin Sensitivity to shear stress: medium |
| Yeast | S.cerevisiae, Pichia pastoris | Production time: Medium Cost: cost of cultivation medium Scalability: highly scalable but high scale-up costs Product yield: High |
Posttranslational modifications: Eukaryotic (can perform O- and N-linked glycosylation) but different from mammalian cells, which may affect the functional activity and decrease half-life of proteins. Sensitivity to shear stress: medium |
| Insect cell lines | Insect-Baculovirus (Sf9, Sf21, Trichoplusia ni) | Product yield: Medium to High, High-level accumulation of recombinant proteins5 Cost: cost of cultivation medium to high, easier to culture than mammals cell lines (more tolerant to changes in osmolarity and by-product accumulation) but complex nutrient requirements. Scalability: highly scalable but high scale-up costs |
Posttranslational modifications: eukaryotic-type posttranslational modifications (including glycosylation) but depends on strain and product Gene size: limited Sensitivity to shear stress: high Production time: Long Safety: Lacks mammalian pathogens but require complex target purification (due to potential contamination with coproduced baculovirus particles) |
| Mammal cell lines | VERO, CHO, MDCK, HEK293, HeLa, NS0, Sp2/0 | Posttranslational modifications: wide variety of posttranslational modifications Product yield: Medium to High |
Gene size: limited Sensitivity to shear stress: high Production time: long, lengthy and laborious process Safety: mammalian pathogens/potential safety risks, including product contamination with residual host cell–derived proteins, mammalian viruses, or mammalian DNA with oncogenic activity Scalability: limited manufacturing capacity, high scale-up costs Cost: cost of cultivation high, bioreactors very expensive to develop and maintain, with complex nutrient requirements, poor oxygen and nutrient distribution, waste accumulation |
| Plant | leaves, tubers, seeds, fruit Tobacco | Posttranslational modifications: eukaryotic posttranslational protein modification machinery (including disulfide bond formation, N-glycosylation substantially similar to that found in mammalian cells even though N-linked glycans terminal residues and the structure of O-linked glycans7 differ between mammalian and plant. Difference in glycosylation patterns may alter the function of the recombinant protein or decrease immunogenicity. Even though glycosylation profiles between animal and plant cells is different, protein folding remains stable Safety: no mammalian pathogens, plant-specific pathogens and viruses do not infect humans. Gene size: unlimited Cost of cultivation: low Scalability: highly scalable at low cost |
Product yield: high but large land requirements, low surface productivity and expensive infrastructure needs. purification of proteins from plants is inconvenient because they cannot be secreted Production time: long (slow growth cycles), Light-dependent production Environment: contamination risk by GMO (risk of gene flow via transgenic pollen) |
| Algae | Chlamydomonas reinhardtii, Volvox carteri, Chlorella sp., Phaeodactylum tricornutum (diatom), Synechococcus, Schizochytrium, D. salina | Posttranslational modifications: eukaryotic posttranslational protein modification machinery. gene expression machinery in chloroplasts is of the prokaryotic type (lacking, for example, glycosylation) but chloroplasts can perform disulfide bond formation9 Production time: Short, minimal nutritional needs. Safety: no mammalian pathogens, toxin free Cost of cultivation: very low Scalability: highly scalable at low cost |
Product yield : Generally low |
The Algae Solution : Microalgae
Microalgae offer several advantages for large-scale production of high-value products:
- Complex post-translational modification pathways.
- Rapid growth cycles with doubling of biomass within 24 hours.
- Safety, with many species considered safe for human consumption (GRAS).
- Low-cost production and reduced downstream processing costs.
- Controlled growth in photobioreactors to prevent environmental contamination.
- Potential use of algal cells as freeze-dried biomass for oral treatments.
Over the past two decades, genetic transformation has been successful in approximately 22 species of microalgae. Notably, Chlamydomonas reinhardtii is the most commonly used algae species for recombinant protein production. All three of its genomes have been sequenced, and molecular tools for genome manipulation have been developed. These advancements have led to the production of enzymes, antigenic peptides, and antibodies, including mAbs.
Challenges and Future Prospects
Despite remarkable progress, several challenges persist in microalgal protein expression systems:
- Lack of standard procedures for genetic transformation of commercially important microalgae species.
- Limited availability of molecular toolkits for genetic engineering.
- Genetic instability and low expression levels of recombinant proteins, which can be addressed with site-directed insertion, inducible promoters, and optimized regulatory sequences.
- In 2016, there were no cGMP industrial-scale algae-based facilities, highlighting the need for further infrastructure development.
Algae-Based Biopharmaceuticals: A Glimpse into the Future
Microalgae are already making their mark in the biopharmaceutical landscape. Several biopharmaceuticals have been produced in algae, including:
- Preclinical vaccines.
- Immunotoxins to fight B-cell lymphomas.
- Antibodies targeting specific diseases.
- Cytokines.
- Utilization of algae as biofactories and delivery vehicles of functional dsRNA.
The road ahead in this field is a promising one, with further research and development poised to unlock the full potential of microalgae as a biopharmaceutical production platform. As we embrace the potential of these tiny aquatic powerhouses, the future of protein expression in biopharmaceuticals looks brighter than ever.
Sources:
- Annual reviews
- Eurekaselect
- Persistent Market Research