Human Practices

Overview

Humans need improved methods for making therapeutic antibodies. Producing these complex proteins is hard but important; the 2014 Ebola Outbreak illustrates the urgency. In response, we propose the use of microalgae as a widescale antibody production platform. Microalgae have all the benefits of higher-level plants but scale faster and are easier to process. Furthermore, from a holistic point of view, microalgae are an ideal chassis; their primary carbon source is CO2 and they are unlikely to harbor mammalian pathogens.

The Need

"I rate the chance of a nuclear war within my lifetime as being fairly low. I rate the chance of a widespread epidemic, far worse than Ebola, in my lifetime, as well over 50 percent." —Bill Gates

With the most recent Ebola Outbreak, over 28 thousand were infected by the deadly pathogen and an estimated 11 thousand died. Meanwhile, a potent anti-Ebola antibody cocktail, ZMapp, was going through preclinical studies. In a study where 18 heavily Ebola infected monkeys treated with ZMapp, all 18 survived, including several in the hemorrhaging stage of the disease. Given the timing and urgent need, ZMapp was approved for use in humans. One of the first patients treated was Dr. Kent Brantly, the American missionary worker who went on to recover from the disease.

When discussing disease outbreaks, the media often makes mention of the vaccine: an antigen—basically a template for pathogen recognition—that provides the body’s immune system with the ability to recognize future infection (“Active Immunity”). Vaccines are ideal for prevention. While they can develop a robust response against a disease, they take several weeks to “train the immune system” and are not ideal in high-risk areas at the moment of onset. We live in a globalized world. Technology and improved infrastructure allow humans to live in incredibly dense concentration, exceeding those of the past. An antigen that takes weeks to develop, weeks to produce, and weeks to “train the immune system” will be insufficient to meet the demands of an emergent virulent pathogen.

Antibodies are unique and powerful tools for eliminating pathogens. They eliminate pathogens, such as viruses, by both neutralization—the hindrance of function by binding to the surface—and complementation—calling in the immune system. Human produced antibodies might target upwards of three million antigens. Additional research, like the high-throughput screening at AbVitro, is making is possible to quickly identify antibody sequences that correspond with pathogen/cancer targeting antibodies. Furthermore, wholly synthetic antibodies might augment number of targetable numbers, with computational modeling making it possible to one day predict antibody sequences that match with emerging pathogens. All of these tools will make the finding of pathogen-targeting antibodies simpler, but will not address the shortcomings in existing production methods.

ZMapp’s utility was hindered by lack of supply, not an apparent inability to neutralize the virus (as illustrated by the Rhesus model). Only 7 doses were available throughout the Ebola Outbreak, despite infection rates in the thousands. Ultimately, it represents a problem, or complete lack thereof, for rapid antibody production capabilities.

A single new Chinese hamster ovary antibody facility, stacked with stainless steel vats, runs in the range of 200 million dollars. Furthermore, the facilities are not modular. They are rigid and highly specialized, built for a particular antibody post-FDA approval (and to scale with its market).

A proposed solution was the tobacco plant. A relatively well-understood and engineered organism, it was the method for making ZMapp. Producers inject plant leaves with agrobacterium containing the DNA for the therapeutic antibody. The plants grow and the antibody is purified from the plant cell lysate. In theory, this is a quick and inexpensive method for rapidly producing lots of antibody, dependent upon arable land rather than high-sterility CHO-vats. In practice, it is not. No tobacco-based antibody has yet to reach market, and ZMapp production was not made rapidly enough to help ease the 2014 Ebola Outbreak. The 2014 Ebola Outbreak was horrific and should be learned from. It was the canary bird in the mine. We live in a smaller world than a century ago, and should consider the cost of not having appropriate treatment infrastructure in place, particularly in case a more contagious pathogen than Ebola emerges in a dense community.

Some Solutions

It might one day be possible to make drugs entirely without cells. Protein-producing gels or other systems could produce drugs economically in vitro without concerning the inherent biological complexity and metabolic needs of living organisms (1,2).

It might, alternatively, be possible to inject the mRNA of a desired antibody directly, thereby offloading the antibody production to the patient rather than delivering a bolus of externally produced antibody. Moderna, for example, has developed synthetic mRNA that codes for polypeptides while avoiding immune-surveillance (and therefore elimination). It’s currently unclear how this mRNA will be targeted to delivery into B cells, and for how long the B cells will continue to produce antibodies. This approach, offloading manufacturing of antibodies to the patient, has been extensively researched in the context of AIDS (5, 6, 7, 8 & 9). Many of these projects are still under development but have fell short for an inability to produce sufficient numbers of antibodies, the rapid mutation of viruses, and the lack of an “off-switch” for antibody production. Future advances may circumvent those problems.

In might be possible to design synthetic bacteria (like Synlogic) for the gut that produce Nanobodies (small enough to be produced in bacteria and lacking complex do-sulfide bonds). These pathogen targeting nanobodies might prove capable of reaching the circulatory system after being turned on by an exogenous transcription factor, circumventing two of the aforementioned problems. However, these nanobodies might still face the complication of improper glycosylation and immune clearance.

Despite the volume of researchers working on futuristic, promising methods for pathogen targeting, existing antibodies are known to work. The issue is therefore less a theoretical scientific endeavor and more of a problem of logistics. And logistically, antibodies from Chinese hamster ovary cells never materialized during the 2014 Ebola Outbreak. So the question becomes: can microalgae produce properly folded antibodies at a high enough concentration and at a cheap enough cost to warrant their use as a widescale antibody production platform? We believe it’s worth further investigation and will present the possible implementation of global microalgae production facilities.

A Green Safety Net

Within each of us (health and medication depending) is an adaptive immune system. A major cell in this system is the B Cell. Immunocompetent B cells are covered in B cell receptors (BCRs). These BCRs respond to non-self antigens. When a foreign antigen binds to a BCR, it activates the B cell to turn into plasma B cells, dedicated producers of specific antibodies against a portion of the antigen that triggered the response.

Some of the plasma B cells enter secondary lymphoid organs, otherwise known as “germinal centers.” Here, B cells undergo rapid mutation so that the selected-for plasma B cell population produces higher affinity antibodies. The resulting plasma B cells begin leave the germinal center to pump out high levels of antibody into the bloodstream. (10)

This is what the human species lacks on a macro-level infrastructure. Hospitals and care-centers act as an innate response, responding in the emergence of a contagion to simply quarantine the sick and provide water. They are not designed with the goal of rapidly adapting and attacking the pathogen. For good reason, drugs for human treatment undergo rigorous FDA review before approval. The average inception to market timeline for an FDA approved drug is 12 years (note: this is after the drug has been developed). This is too long for rapid drug turnaround and represents a significant threat to human health.

Microalgae as a production platform would represent an adaptation of existing production systems. Commercial antibodies are produced after recombinant DNA is inserted into Chinese hamster ovary (CHO) cells. The cells are grown in growth media, the cells are lysed and the antibody is purified by Protein A resin. The volume of available steel vats determines the quantity of CHO cells and therefore protein. Microalgae represent an ideal solution. They posses the machinery for disulfide bonds, are large enough to produce antibodies without inclusion bodies, and can properly glycosylate; in fact, their glycoproteins are considered similar to mammals’ (3).

As such, microalgae “race track ponds,” (such as those imaged below) could be used to produce enormous quantities of antibody quickly. The ponds require only inexpensive media, like filtered salt-water with added ammonium, along with CO2 and environmental protection.

Culture scale-up would allow for controlled growth of the algae from a smaller to a larger batch. Final large-scale ponds could hold volumes in the millions of liters, vs. thousands of liters for conventional CHO facilities. Racetrack ponds have been extensively researched in the context of biofuels. Unfortunately, current levels of lipid production do not reach an economically viable level (i.e. one in contention with the extremely low margins of oil and natural gas industries)(4). However, in higher value markets, such as therapeutics (especially in the potentially subsidized environment of pandemic prevention) the economics could work. Furthermore, large and high throughput microalgae facilities would act as a species-wide adaptive immune response, fulfilling the unmet need for high-volume antibody production.

Cost/Benefit

The true benefit of therapeutic antibodies from microalgae expression systems is their fixed costs. The cost to build the facilities is dramatically less than that of CHO facilities. They will not be more efficient than CHO facilities in the short-term, but will, due to enormous relative volume, be able to match production output (even before doubled effort into genetically engineering of the microalgae). Before and after their use for use making pathogen-treating antibodies, microalgae facilities could be used to produce vitamins, food stock for animals in undeveloped countries, or simply to reduce CO2 from nearby coal and natural gas factories. The coupling of microalgae facilities to the waste flue gas of powerplants has been researched and implemented by companies like Sapphire Energy, Sunomix, and Solazyme.

In industrialized countries with significant carbon taxes, like the UK and Australia, microalgae facilities might become commonplace alongside power plants. Sapphire Energy, Sunomix, and Solazyme have demonstrated the market potential of microalgae products. It would be strategic to further distribute algae plants near heavily populated areas. Were a pandemic to occur, these facilities could expunge their commodity-producing microalgae and replace it with a high antibody producing strain of microalgae, like recombinant Chalmydomonas reinhardtii or Phaeodactylum tricornutum.