On-Demand Antivenom Therapeutics

Imagine walking along a reedy path when suddenly you feel something thump against your leg. You look down to see a saw-scaled viper quickly slithering away. Your leg throbs, and warmth radiates from two puncture wounds on your calf. You've been bitten by a deadly snake. Time is running out, but there might be a treatment. The only hospital in your province, hours away, may not even have it because companies have stopped producing it. Still, you have no choice but to try.

Envenomation, the exposure to toxins from venomous animals like snakes and scorpions, is a global problem that primarily affects people in less developed countries. Snakebites are the most significant form of envenoming, with 1.8 to 2.7 million cases annually. Out of these, approximately 138,000 are fatal, and 400,000 result in permanent disabilities. As a result, the World Health Organization has recognized envenoming as one of the Neglected Tropical Diseases (NTDs), with snakebite-related morbidity and mortality ranking higher than other well-known NTDs. In fact, the burden of snakebite-induced deaths and disabilities in West Africa alone surpasses that of all other NTDs worldwide. Envenomation, particularly affecting children in impoverished countries, is widely regarded as one of the world's most neglected NTDs.

While physicians and health policymakers are well aware of the impact of this global affliction, preventing envenomation-related deaths and disabilities is a challenging task, not due to the lack of effective treatments, but due to logistical and economic obstacles. Venom consists of a potent mixture of poisons and toxins that cause cytotoxicity, hemolysis, and neurotoxicity. However, these venom toxins can be neutralized proteins called antivenoms. When administered, antivenom binds to and sequesters the toxins, minimizing tissue damage and preventing loss of life. Antivenoms are created by injecting animals with small doses of venom to stimulate the production of antibodies. The animal serum is then extracted and purified to create the antivenom therapeutic, which is stored until needed for envenomation cases.

Despite their efficacy, there is currently a severe shortage of antivenoms worldwide, reaching crisis levels. Desperate measures, such as rationing, reducing dosages, and extending expiration dates, are being taken due to the scarcity. The complex and costly process of antivenom biomanufacturing, combined with the small global market in impoverished countries, has created few economic incentives for companies to invest in antivenom production. A recent example is Sanofi's decision to discontinue the production of Fav-Afrique, the only proven antivenom for treating snakebites in sub-Saharan Africa. The company cited manufacturing costs as the main reason for this move. A significant cost factor is the perishability of antivenoms. Once manufactured and processed, they must be stored between 2-8 degrees Celsius, requiring a cold-chain distribution network and reliable cold storage at the destination. This poses a significant challenge for poorer countries, where the need for antivenom is most critical. Furthermore, antivenoms typically have a shelf-life of only 36 months.

Our solution tackles many of these challenges by adopting a novel approach to therapeutic development that flips traditional biomanufacturing on its head.

Our solution addresses the envenomation problem by introducing a groundbreaking platform that revolutionizes antivenom biomanufacturing. Rather than following the traditional approach of producing antivenom in a centralized lab and transporting it under cold storage conditions, we propose a concept called "distributed biomanufacturing." With this approach, the therapeutic is manufactured on-site at the time of administration, eliminating the need for cold chain logistics. Our system is shelf-stable and operates using cell-free biomanufacturing technology. Typically, biomanufacturing involves using animals or cells to produce proteins, which are then processed for purification. In our cell-free system, we extract the protein-synthesizing machinery from cells and utilize it as a biochemical soup. By introducing specific DNA sequences into this cell-free soup, we can generate a wide range of proteins on-demand. A key advantage of our cell-free reactions is that they can be freeze-dried for stable storage at room temperature. By rehydrating the freeze-dried cell-free (FDCF) reaction with water and the appropriate DNA program, we can fully synthesize the desired protein therapeutic quickly, within 1-2 hours. This protein can then be purified using a custom hand-held device and the final therapeutic is then ready to use.

Essentially, our on-demand production system mirrors the flexibility of freeze-dried systems like M.R.E.s (meals-ready-to-eat), but specifically for emergency therapeutics. In a peer-reviewed research study, we have demonstrated the efficiency and versatility of these FDCF reactions by successfully producing 10 peptide antibiotics, 5 enzymes, 4 protein vaccines, and 15 different antibodies. (For our paper, see DOI: 10.1016/j.cell.2016.09.013). By applying this technology to the production of antibodies used as antivenoms, we can overcome several limitations inherent in the inefficient conventional centralized biomanufacturing paradigm. Our approach eliminates current chokepoints and streamlines the production process, paving the way for a more effective and accessible antivenom supply.

The implementation of a distributed biomanufacturing approach offers numerous benefits in the production and distribution of antivenoms. Firstly, it eliminates the need for cold chain distribution and maintenance of antivenoms, making transportation simpler and enabling low-resource medical facilities to access this life-saving therapy. Instead of stocking various antivenoms, on-site production from a DNA library can be carried out when needed, greatly reducing waste and ensuring the availability of the required therapeutic. Moreover, this approach leads to significant cost-savings compared to the current centralized infrastructure. Cold chain circumvention and on-demand production minimize embedded costs in centralized production, processing, and distribution. Producing FDCF reactions, derived from extracts or specific cell components, is relatively inexpensive, estimated at $10 to produce approximately 10 mg of antivenom, assuming a fair production yield. Furthermore, distributed biomanufacturing also contributes to a reduction in energy usage and environmental footprint. The elimination of refrigeration requirements and the removal of around 99% of the weight (typically water) through freeze-drying simplify shipping processes and further minimize the environmental impact. In summary, implementing distributed biomanufacturing offers simplified access, reduced waste, cost-savings, and environmental benefits for the production and distribution of antivenoms.

Our proposed distributed biomanufacturing of antivenoms, using the FDCF (Freeze-Dried Cell-Free) approach, aims to significantly improve the lives of billions of people at risk of snake and insect envenomation. These individuals primarily reside in poorer countries located in the tropical and subtropical regions of Africa, Asia, and Latin America. Annually, there are 2.7 million cases of snakebite envenomations resulting in death and disability. Children in these countries face a particularly high risk, as snake encounters often occur during play and exploration. Given their smaller body size, snakebites can be even more lethal for children. Envenomation also disproportionately affects rural communities, especially those in the agricultural sector, who are highly exposed to venomous animals and may have limited access to medical care.

To ensure the success of our on-demand antivenom solution, we will actively engage with key communities who would be the end-users of our technology. We aim to integrate their insights and feedback into the design and development process. Specifically, we seek to gain the following:

1. Insight from end users, including rural doctors, community leaders, and those at high risk of envenomation, regarding our unique distributed biomanufacturing approach. We value their feedback to improve the practical implementation of our solution.
2. Understanding the specific environmental resources and user interface requirements necessary for reliable execution of the FDCF reactions and subsequent purification module. We aim to assess whether our system can be utilized beyond hospitals, such as in rural villages.
3. Mapping out local regulatory and clinical safety hurdles that may need to be addressed at a fundamental level within our system. Additionally, we aim to determine the clinical trial requirements for approval of this novel approach as a viable therapeutic modality.
4. Assessing the variations in these factors across major global regions to determine if a single platform can meet diverse requirements.

Obtaining the Horizon Prize is crucial for driving our outreach initiative and involving these end users in the design-build-test collaboration cycle. We have laid the foundation for bringing affordable, effective, and rapid antivenoms to the poorest areas of the world. We firmly believe that our technology has the potential to save millions of people from the tragic consequences of envenomation, such as permanent injuries or death. However, to make the technology truly effective and practical, we recognize the necessity of incorporating the guidance and insights of the target communities into our development process.

Our team primarily consists of academics, including scientists and engineers, who stumbled upon a groundbreaking method for rapidly manufacturing proteins using freeze-dried cellular extracts. We recognize that these FDCF reactions have immense potential for manufacturing therapeutics and can bring biomanufacturing to those who need it most. Our goal is to democratize biotechnology, which has traditionally been limited to wealthier nations. To achieve this, we have expanded our collaboration network to include the BioMANufacturing Consortium at MIT and engaged with researchers residing in the target regions, who have expressed excitement at the potential for the FDCF-based antivenoms.

Admittedly, our current team does not yet fully represent the communities we aim to serve. However, we are driven by our passion to use this technology to revolutionize the production and accessibility of therapeutics in impoverished countries. We understand the necessity of expanding our team to include global representatives from these communities at various levels, including local scientists, NGOs, clinical leaders, town/village coordinators, and the end-users of antivenoms. By incorporating these diverse perspectives, we can refine the antivenom production system to ensure practical implementation that meets the needs of all stakeholders.

The Horizon Prize funds will play a crucial role in supporting our efforts, enabling us to include representatives from these communities and gather their invaluable input. With their contributions, we can optimize the antivenom production system and work towards our shared goal of making a positive impact on the lives of those in need of ubiquitous antivenom access.

Our technology's core component has undergone comprehensive development and testing over multiple years. Through rigorous testing, we have successfully demonstrated the capabilities of our refined FDCF on-demand system in producing various protein therapeutics, including vaccines, enzymes, and antibiotic peptides. Notably, our system excels in generating antibodies, the essential molecules comprising antivenoms. In order to advance to the pilot stage, our next objective is to design a front-end device that enables the implementation of our FDCF reactions in real-world settings. For detailed information on the development of our FDCF system, please refer to our manuscript titled 'Portable, On-demand Biomolecular Manufacturing'.

The Horizon Prize presents a tremendous opportunity to propel our antivenom biomanufacturing project forward, transitioning from a proof-of-concept technology to the development of a practical prototype. We have identified several key challenges that we aim to address through the support of the Prize:

1. Supporting Personnel: Securing significant financial backing is crucial for advancing this endeavor. Envenomation, although a significant neglected tropical disease, is not considered a profitable market, making it challenging to find adequate financial support. The Horizon Prize would not only provide the necessary financial resources for our team to dedicate their time to this project but also increase the visibility of our innovative approach, potentially attracting additional funding from other organizations.
2. Addressing Development Barriers: We recognize the importance of expanding our collaboration to include members of the target communities. To achieve this, a portion of the funds will be allocated to develop a global network, enabling us to ship key prototypes to low-resource settings for testing. This will allow us to establish critical optimization parameters for our distributed envenomation biomanufacturing platform. The funding will support the acquisition of communication equipment and field data logging tools to facilitate effective collaboration.
3. Tackling Technological Barriers: The foundational FDCF reactions, enabling rapid on-demand protein production, have been fully demonstrated and developed. However, we still need to design and develop the downstream processing device responsible for achieving the required purity of the antivenom for administration. It is essential that this device is user-friendly, robust, and suitable for practical field use without relying on specialized training. The insights and feedback from our global collaborators will inform the device's intuitive design. The Horizon Prize will fund personnel efforts and materials necessary for the development of this purification module.
4. Overcoming Regulatory Barriers: Given the significant departure from traditional centralized manufacturing, we anticipate challenges in obtaining regulatory approval for our on-demand manufactured antivenom. Furthermore, the processing device will need to demonstrate consistent purification of the antivenom, address contamination prevention, and undergo rigorous safety testing. The novel nature of our technology and its intended use in the field will undoubtedly attract higher scrutiny. Here, the Horizon Prize will support our clinical trial efforts, third-party safety assessments, and regulatory compliance fees.

By addressing these personnel, development, technological, and regulatory barriers, the Horizon Prize will provide the essential resources and support needed to advance our antivenom biomanufacturing project, bringing us closer to our goal of providing affordable and accessible antivenom to those in need.

As a Research Scientist at the Wyss Institute of Biologically Inspired Engineering, the Team Lead plays a crucial role in a global network that connects academic research with the goal of addressing the snakebite envenomation crisis. The primary focus of the Team Lead lies in developing this technology for practical implementation, with a emphasis on establishing strong connections to the communities in need. At the Wyss Institute, our overarching mission is to engineer biologically inspired materials and devices that have the potential to revolutionize healthcare and promote sustainability. In order to achieve this, we strive to foster strong connections with the communities that would benefit from our antivenom platform.By listening to their insights and involving them in the design and implementation process, we aim to create a truly impactful and community-driven solution.

Our solution represents not only a groundbreaking approach to manufacturing antivenoms, but also a completely new paradigm in biomanufacturing as a whole. Instead of relying on centralized locations and refrigeration to produce and store therapeutics, which often results in significant waste and high costs, our shelf-stable FDCF technology allows for the on-demand production of any protein-based therapeutic precisely when it's needed. The DNA responsible for directing the production of specific proteins is also freeze-dried, further simplifying the process. The only additional resource required, which can be easily provided at the point of use, is sterile water. Moreover, refrigeration becomes unnecessary as the therapeutic can be administered shortly after production. This approach has the potential to revolutionize therapeutic biomanufacturing, particularly in resource-limited settings.

• Impact goal for next year - complete development of the FDCF device module for field use. Begin organizing a global network of trial test centers that focus on local end-user (non-hospital) testing. All of this will require substantial funding, so grant and non-profit funding sources are essential in making this effort viable.
• Impact goal for five years - become a visible leader for an alternative approaches to the envenomation crisis. At this point, we would have engaged with the local target regions to establish a timeline for regulatory approval. This would require not only additional funding, but also higher visibility to introduce our paradigm-shifting approach to therapeutics manufacturing and distribution.

We have completed Phase I of our technical development objectives, which was focused on developing and testing our freeze-dried cell-free system. We have tested the breadth of proteins the system can produce, the yields, and also performed animal testing to demonstrate its safety and efficacy.

We have started Phase II of our technical development, which is the device development for controlling the FDCF reactions and purifying the resulting antivenom. Part of this process involves screening various published antivenoms to establish the ones that would optimally suit the parameters of our platform and the intended region we would like to trial the system in.

Contacts in our target trial countries is one key indicator we are currently trying to improve. Developing different levels of contacts in these regions is a critical goal for establishing the clinical test beds and contacts for design feedback. Here, the indicators we wish to fill are sufficient contacts at these levels with 'buy-in' for testing our system. 

Our technology solves the following major bottlenecks in current antivenom production that has led to the current crisis:

• By eliminating the need for cold chain distribution and maintenance of antivenoms, we will greatly simplify current transportation complexities and enable low-resource medical facilities to access this life-saving therapy.  
• Implementing a distributed biomanufacturing approach would greatly reduce waste of the therapeutics, as the required therapeutic could be made on site when it is needed from a library of DNA rather than requiring a stock of various antivenoms to be maintained on site.
• Lastly, a significant reduction in energy usage and thus environmental footprint would be achieved in that no refrigeration is required and that the freeze-dried reactions would have approximately 99% of the weight (which is typically water) removed, simplifying shipping.

• Cold chain circumvention and the on-demand nature of production would result in a drastic cost-savings in comparison to the current infrastructure, where much of the cost is embedded in the centralized production, processing, and distribution. The FDCF reactions themselves, derived from extracts or specific components of cells, are inexpensive, with an estimated cost of $10 to produce ~10 mg of antivenom, assuming a fair production yield.
• Point-of-care production - by enabling the on-demand production of therapeutics at the point of administration, we have the potential to democratize biotechnology. This approach eliminates a significant portion of the costs associated with manufacturing the therapeutic. While this may impact the profit margin of traditional biotechnology companies that rely on centralized manufacturing, our approach empowers the local community, which is directly affected by biomanufacturing, by giving them control over the process.

We have a simple, portable platform that enables the on-site and on-demand manufacturing of therapeutics and biomolecules. This flexible system utilizes reaction pellets made of freeze-dried, cell-free transcription and translation machinery. By adding DNA encoding the desired output and hydrating the pellets, protein biomanufacturing can be easily achieved. In a nutshell, our technology is 'just-add-water therapeutic production'.

We have demonstrated the use of our FDCF platform for the rapid production of 10 peptide antibiotics, a biosynthetic pathway composed of 5 enzymes that makes antivirals, 4 protein vaccines (verified in mice), and 15 different antibodies. Our manuscript on this work was published in Cell, and can be found here: 

https://www.cell.com/cell/full...

Portable, On-Demand Biomolecular Manufacturing
Pardee, Keith et al.
Cell, Volume 167, Issue 1, 248 - 259.e12

Three full-time staff.

Since 2016, so approximately 7 years.

Our small team includes a female scientist and at our core, we are deeply committed to fostering diversity, equity, and inclusion in everything we do. We firmly believe that diverse perspectives, backgrounds, and experiences drive innovation and enable us to better serve the communities we aim to impact.

We hope to expand our global test network soon to include researchers and end-users from the key target countries, which encompass low-income regions in Asia, Latin America, and Africa. For our on-demand antivenom to truly realize its potential, it will require a collaborative effort - expanding our team to include those that are marginalized, who are the key population who would benefit from our technology.