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.