Expression and manufacturing of protein therapeutics in spirulina

Jester, Benjamin W.; Zhao, Hui; Gewe, Mesfin; Adame, Thomas; Perruzza, Lisa; Bolick, David T.; Agosti, Jan M.; Khuong, Nhi; Kuestner, Rolf E.; Gamble, Caitlin; Cruickshank, Kendra; Ferrara, Jeremy; Lim, Rachelle; Paddock, Troy; Brady, Colin J.; Ertel, Stacey; Mia, Zhang; Tasch, Michael; Saveria, Tracy; Doughty, D.; Marshall, Jacob; Carrieri, Damian; Lee, Jamie; Goetsch, Lauren; Dang, Jason; Sanjaya, Nathaniel; Fletecher, David; Martinez, Anissa; Kadis, Bryce; Sigmar, Kristjian; Afreen, Esha; Nguyen, Tammy; Randolph, Amanda; Taber, Alexandria; Krzeszowski, Ashley; Robinett, Brittney; Grassi, Fabio; Guerrant, Richard L.; Spigarelli, Michael G.; Takeuchi, Ryo; Finrow, Brian; Behnke, Craig A.; Roberts, James

doi:10.1101/2021.01.25.427910

Cited by 8 publications

(15 citation statements)

References 68 publications

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“…Using western blot, we found that the GFP fusion protein comprised 2.7% of total protein ( Supplemental Figure S1 ). This level was within the typical range for antibodies in development as therapeutics 7 .…”

Section: Resultssupporting

confidence: 64%

“…These include simple, inexpensive growth and downstream processing; photosynthetic metabolism; and Generally Regarded as Safe (GRAS) status with the FDA [4][5][6] . Genetic engineering of spirulina allows for stable expression of heterologous proteins, including diverse anti-pathogen proteins such as active vaccine antigens, antibodies, and therapeutic enzymes 7 . Together, these features offer the potential to produce disruptively low-cost biologics and biologic cocktails.…”

Section: Introductionmentioning

confidence: 99%

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Machine Learning Optimization of Photosynthetic Microbe Cultivation and Recombinant Protein Production

Gamble¹,

Bryant

Carrieri

et al. 2021

Preprint

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Background: Arthrospira platensis (commonly known as spirulina) is a promising new platform for low-cost manufacturing of biopharmaceuticals. However, full realization of the platform's potential will depend on achieving both high growth rates of spirulina and high expression of therapeutic proteins. Objective: We aimed to optimize culture conditions for the spirulina-based production of therapeutic proteins. Methods: We used a machine learning approach called Bayesian black-box optimization to iteratively guide experiments in 96 photobioreactors that explored the relationship between production outcomes and 17 environmental variables such as pH, temperature, and light intensity. Results: Over 16 rounds of experiments, we identified key variable adjustments that approximately doubled spirulina-based production of heterologous proteins, improving volumetric productivity between 70% to 100% in multiple bioreactor setting configurations. Conclusion: An adaptive, machine learning-based approach to optimize heterologous protein production can improve outcomes based on complex, multivariate experiments, identifying beneficial variable combinations and adjustments that might not otherwise be discoverable within high-dimensional data.

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Section: Resultssupporting

confidence: 64%

Section: Introductionmentioning

confidence: 99%

Machine Learning Optimization of Photosynthetic Microbe Cultivation and Recombinant Protein Production

Gamble¹,

Bryant

Carrieri

et al. 2021

Preprint

Self Cite

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“…Genes encoding pp1005, pp1006, pp1007, pp1093 and pp1092 were engineered into the spirulina chromosome by homologous recombination (Figure 1B) 45 . The final strains—SP1308, SP1312, SP1313, SP1286 and SP1287, respectively—contained the gene of interest integrated into the spirulina chromosome at a single, pre-selected location and no other exogenous genetic information.…”

Section: Resultsmentioning

confidence: 99%

“…LMN-201 was manufactured using the edible microorganism spirulina ( Arthrospira platensis ) as the expression platform. It was delivered orally as a powder of whole, unrefined spirulina biomass 45 , and is easily scalable, shelf-stable, simple to administer, and broadly neutralizing against all common TcdB types.…”

Section: Introductionmentioning

confidence: 99%

Using synthetic activity to design ultra-potent antibody cocktails

Zhao¹,

Tasch²,

Dodds³

et al. 2021

Preprint

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We applied a mathematical framework originally used to model the effects of multiple inhibitors on enzyme activity to guide the development a therapeutic antibody cocktail, LMN-201, to prevent and treat C. difficile infection (CDI). CDI causes hundreds of thousands of cases of severe, often recurrent diarrhea and colitis in the United States annually and is associated with significant morbidity and mortality worldwide. Current therapies for preventing recurrent CDI are only partially successful, and there are no options available to prevent initial bouts of CDI in at-risk populations. Almost all antibody therapies have been developed and administered as monotherapies. Antibody cocktails are relatively rare even though they have the potential to greatly increase efficacy. One reason for this is our limited understanding of how antibody interactions can enhance potency, which makes it difficult to identify and develop antibodies that can be assembled into optimally effective cocktails. In contrast to the view that antibody synergies depend on unusual instances of cooperativity or allostery, we show that synergistic efficacy requires nothing more than that the antibodies bind independently to distinct epitopes on a common target. Therefore, synergy may be achieved much more readily than is generally appreciated. Due to synergy the LMN-201 antibody cocktail, which targets the C. difficile exotoxin B (TcdB), is 300- to 3000-fold more potent at neutralizing the most clinically prevalent TcdB toxin types than bezlotoxumab, the only monoclonal antibody currently approved for treatment or prevention of CDI. The efficacy of LMN-201 is further enhanced by inclusion of a phage-derived endolysin that destroys the C. difficile bacterium, and which therefore has a complementary mechanism of action to the antibody cocktail. These observations may serve as a paradigm for the development of high potency biologic cocktails against targets that have proven challenging for single-agent therapies.

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“…19 One non-traditional platform being pursued (at least for single chain antibody production) is Spirulina. 76 Alternative strategies such as transgenic animals that express human MAbs including human IgA in colostrum and breast milk are also worthy of investigation, especially considering their track record with other biologics. 77,78 While oral administration of MAbs and MAb cocktails has obvious benefits over intravenous and subcutaneous routes of delivery, the pharmacokinetics and stability of SIgA in the human gastric and intestinal environments remain unknown and need to be taken in consideration.…”

Section: Summary and Perspectivesmentioning

confidence: 99%

The prospect of orally administered monoclonal secretory IgA (SIgA) antibodies to prevent enteric bacterial infections

Richards

Baranova

Mantis

2021

Human Vaccines & Immunotherapeutics

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Eliminating diarrheal diseases as a leading cause of childhood morbidity and mortality in low-and middleincome countries (LMICs) will require multiple intervention strategies. In this review, we spotlight a series of preclinical studies investigating the potential of orally administered monoclonal secretory IgA (SIgA) antibodies (MAbs) to reduce disease associated with three enteric bacterial pathogens: Campylobacter jejuni, enterotoxigenic Escherichia coli (ETEC), and invasive Salmonella enterica serovar Typhimurium. IgA MAbs targeting bacterial surface antigens (flagella, adhesins, and lipopolysaccharide) were generated from mice, humanized mice, and human tonsillar B cells. Recombinant SIgA1 and/or SIgA2 derivates of those MAbs were purified from supernatants following transient transfection of 293 cells with plasmids encoding antibody heavy and light chains, J-chain, and secretory component (SC). When administered to mice by gavage immediately prior to (or admixed with) the bacterial challenge, SIgA MAbs reduced infection C. jejuni, ETEC, and S. Typhimurium infections. Fv-matched IgG1 MAbs by comparison were largely ineffective against C. jejuni and S. Typhimurium under the same conditions, although they were partially effective against ETEC. While these findings highlight future applications of orally administered SIgA, the studies also underscored the fundamental challenges associated with using MAbs as prophylactic tools against enteric bacterial diseases.

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Expression and manufacturing of protein therapeutics in spirulina

Cited by 8 publications

References 68 publications

Machine Learning Optimization of Photosynthetic Microbe Cultivation and Recombinant Protein Production

Machine Learning Optimization of Photosynthetic Microbe Cultivation and Recombinant Protein Production

Using synthetic activity to design ultra-potent antibody cocktails

The prospect of orally administered monoclonal secretory IgA (SIgA) antibodies to prevent enteric bacterial infections

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