Development of spirulina for the manufacture and oral delivery of protein therapeutics

Modern biotechnology relies on the domestication of cells as biological factories through genetic engineering. Expression platforms include *Escherichia coli*, used to manufacture relatively small and simple therapeutic proteins, and yeasts and mammalian cells for more complex molecules. Adoption of new expression platforms depends on the availability of methods for genetic manipulation of the organism to achieve stable, high expression of exogenous proteins, and on whether the organism possesses biological traits compatible with large-scale manufacturing and commercialization. Genetically engineered plants have performance characteristics different from cultured cells, such as photosynthetic growth and easy scalability. However, their promise has not been realized for reasons including cumbersome genetic methods, slow growth rates, low product yields and regulatory constraints. Algae have been considered as alternatives to plants for biotechnology applications, but are difficult to engineer genetically and expression levels of exogenous protein are low and often unstable. To date, no biologic therapeutic has been commercialized using an algal platform. Photosynthetic spirulina is the only microorganism that is commercially farmed worldwide as a food. Its protein content exceeds that of all other food crops, making it a strong candidate for the expression of therapeutic proteins at high levels. Spirulina’s asexual reproduction mitigates the risk of gene escape into the food chain and the associated food security concerns and regulatory burden. Spirulina therefore promises the benefits of plant-based biopharmaceuticals and may overcome the challenges and limitations of other crop- and algal-based platforms.

This section of the paper reviews existing literature on various biomanufacturing platforms, highlighting their strengths and limitations. The authors discuss the use of *E. coli*, yeasts, and mammalian cells, noting the challenges associated with scaling up production and the limitations of each system in terms of the complexity of proteins they can effectively produce. The use of genetically engineered plants is also explored, with a focus on the difficulties related to genetic manipulation, slow growth rates, and regulatory hurdles. Finally, the authors examine previous research on algal platforms, acknowledging the challenges of genetic engineering and low/unstable expression levels in algae. This comprehensive review sets the stage for the introduction of spirulina as a potential alternative, emphasizing its unique advantages.

This research employed several key methodologies: **Genetic Engineering of Spirulina:** The researchers developed novel genetic engineering methods for *Arthrospira platensis* (spirulina). This involved demonstrating that spirulina is naturally competent for transformation, a previously unrecognized trait. Transformation was achieved through co-cultivation with companion microorganisms (*Sphingomonas* and *Microcella*), which induced competence. The study employed markerless homologous recombination for the stable integration of exogenous genes into the spirulina chromosome, achieving high-level expression of diverse proteins. **Protein Expression and Characterization:** A variety of exogenous proteins were expressed in spirulina, including bioactive peptides, single-chain antibodies (VHHs), enzymes, and vaccine antigens. Different scaffolding strategies were used to optimize VHH expression and avidity, including monomers, dimers, trimers, and heptamers. The expression levels of these proteins were quantified, and their bioactivity was confirmed through various assays. **Large-Scale Cultivation:** The researchers developed and optimized a large-scale, indoor cultivation system for spirulina using modular, vertical, flat-panel photobioreactors. This system allowed for controlled growth under current good manufacturing practices (cGMP) conditions, addressing the limitations of open-pond cultivation for biopharmaceutical production. **Downstream Processing:** Harvested spirulina biomass was processed through a simplified downstream process involving rinsing with trehalose solution and spray-drying. The researchers optimized spray-drying parameters to maximize efficiency and maintain protein activity. The resulting dry powder was stable without refrigeration, facilitating storage and distribution. Finally, the dry powder was encapsulated into vegetarian capsules for oral delivery. **In Vitro and In Vivo Studies:** In vitro studies assessed the stability of the expressed proteins under simulated gastric and duodenal conditions. In vivo studies using mouse models of *Campylobacter jejuni* infection demonstrated the efficacy of orally delivered spirulina-expressed VHHs in preventing disease. The researchers measured fecal shedding of *C. jejuni*, markers of intestinal inflammation, and assessed the impact of treatment on *C. jejuni* motility. **Clinical Trial:** A phase 1 clinical trial evaluated the safety and tolerability of spirulina-expressed VHHs in healthy human volunteers. The study assessed adverse events, laboratory abnormalities, and VHH serum levels.

The key findings of this study are: 1. **Spirulina's Natural Competence:** The authors discovered that *Arthrospira platensis* exhibits natural competence for transformation, overcoming a significant barrier to genetic manipulation. This was further enhanced through co-cultivation with specific companion microorganisms. 2. **High-Level, Stable Protein Expression:** They developed methods for stable, high-level expression of diverse therapeutic proteins in spirulina, with expression levels reaching up to 29% of total soluble protein. This was achieved through markerless homologous recombination and the use of strong constitutive promoters. 3. **Multimeric VHH Scaffolding:** The use of various scaffolding strategies allowed the efficient production of multimeric VHHs with subnanomolar apparent KD levels. This improved binding avidity, potentially accelerating product development. 4. **Large-Scale cGMP Production:** A large-scale, indoor cultivation system using modular photobioreactors was developed and optimized for cGMP-compliant production. This eliminates the contamination risks associated with open-pond systems. 5. **Simplified Downstream Processing:** The simple downstream process, involving spray-drying and encapsulation, is cost-effective, maintains protein activity, and eliminates the need for extensive purification before oral delivery. 6. **Gastric Protection and Oral Delivery:** Encapsulation within dry spirulina biomass protects the therapeutic proteins during gastric transit, ensuring delivery to the target site in the intestine. A modified VHH demonstrated increased resistance to proteases. 7. **In Vivo Efficacy:** Oral delivery of spirulina-expressed anti-*Campylobacter* VHHs significantly reduced *C. jejuni* infection and inflammation in mouse models. 8. **Phase 1 Clinical Trial Success:** A phase 1 clinical trial demonstrated the safety and tolerability of the spirulina-based therapeutic product (LMN-101) in healthy human volunteers.

This study successfully addresses the need for cost-effective and scalable production platforms for oral biologics. The authors demonstrate that spirulina, a safe and readily available food source, can be efficiently engineered to produce high yields of diverse therapeutic proteins. The simplified downstream processing and the stability of the product without refrigeration represent significant advantages over existing platforms, particularly for applications in developing countries. The in vivo and clinical trial data support the potential of spirulina-based therapeutics for the prevention and treatment of infectious and other diseases. The platform's versatility and scalability suggest its broad applicability to a wide range of therapeutic proteins, potentially transforming the development of complex multicomponent biologic cocktails.

This research demonstrates a novel and highly promising platform for the cost-effective and scalable production of orally delivered protein therapeutics. The successful genetic engineering of spirulina, coupled with its inherent safety and simplified manufacturing process, provides a significant advancement in the field. Further research should focus on expanding the range of expressible proteins, optimizing cultivation and downstream processing, and conducting larger clinical trials to assess efficacy across various disease indications.

While the study demonstrates significant promise, there are some limitations. The efficacy of the spirulina-expressed VHHs was primarily demonstrated in mouse models of *Campylobacter jejuni* infection; further investigation is needed to confirm efficacy in humans. The study focused on a specific VHH; additional research is needed to explore the applicability of the platform to a broader range of therapeutic proteins. The long-term stability of the product under various storage conditions requires further evaluation. Finally, the dependence on companion microorganisms for transformation may require further optimization for robust large-scale cGMP manufacturing.