Claudia Jimenez, General Manager of ALGENEX

“We are years ahead of any competitor that may appear in the future,” says Claudia Jimenez, General Manager of biotech company ALGENEX, which uses moths to develop vaccines.

What was the original inspiration for deploying living organisms in the production of recombinant protein? Why was there a need and demand for an ‘alternative form’ of gene expression?

Biologics have traditionally been manufactured using bioreactors. These facilities, which require a significant amount of capital expenditure to construct, have significant limitations in terms of scalability, versatility, yield and cost. This has led to a number of research groups to develop alternative systems for protein expression using living organisms such as green plants or transgenic mammals. The whole organisms contain living cells in perfect physiological conditions, in contrast to cultured cells which depend on synthetic artificial media quality, gases concentrations, sterility conditions and many other factors that may affect the productivity and safety of the system. In summary, if you develop a method to programme the cells in a living organism, you get a more simple and robust system to produce recombinant biologics.

We understand that ALGENEX initially started out by trying to harness transgenic green plants to develop vaccines, but subsequently opted to use insects instead of plants. What prompted this shift of approach?

Indeed, in the 90s the founder’s research group started experimenting with green plants, in parallel to the pioneers in this idea. But based on the low expression yields obtained with transgenic plants, the scientific team reached the decision of evaluating alternatives. An international collaboration allowed the group to verify the power of insects as protein expression tool, which in turn led to the founding of Algenex. Additionally, insects contain a higher percentages of protein in comparison to green plants, with insect cells being champions in protein production, also in terms of scalability. From just a couple of moths, in nine weeks it is possible to obtain 250 millions of individuals. Recently, a company in Denmark claimed to produce 1,2 Tn of insect biomass daily for protein additives to animal food, but in the future they want to produce more than 100 Tn per day. No other organisms may produce at this scale in a small space and so fast.

Tell us more about the science behind using the baculovirus to infect insects and the process of employing caterpillar pupae as a biological factory for cheaper and easier recombinant protein manufacturing.

The technology of using of baculovirus to programme cultured insect cells to express proteins has been around for over 30 years. In fact, the technology was patented in 1988 by Smith & Summers under US Patent No. 4,745,051: Method for Producing a Recombinant baculovirus Expression Vector. It is well known that transient expression (Baculovirus vectors) significantly reduces development time and associated costs while increasing versatility and maintaining the biological safety of the products.

Seeking an alternative to BEVS-mediated production in bioreactors, Algenex chose to develop a platform using the Trichoplusia ni Lepidoptera, which is the insect host for the AcMNPV vector, the Baculovirus most widely used in the industry. First studies infecting directly the insect larvae showed evidence of the advantages of the technology in terms of flexibility, scalability, yield and cost. This Baculovirus produces a lethal lytic infection in nature. It means that the genetically modified vector we prepare in the laboratory will infect practically all the insect cells. As soon as the cell is infected, it begins to produce the desired protein (encoded by the vector genome), and in a few days, the peak of productivity is reached, and it is the moment to collect the protein from the insect biomass by simple and conventional procedures. Nature developed the tools, and by simple genetic manipulation of a common virus vector totally harmless for mammals, we obtain the complex product. This process can be achieved in the larval or pupae stages. In Algenex we work with insect pupae for many reasons, which include process automation, storage and transport conditions of the insect and simplicity in technology transfer. In many aspects, the production in pupae resembles the vaccines production in eggs. However, the production in pupae is much more flexible and scalable, producing any recombinant protein product encoded by the widely used baculovirus vector derived from AcMNPV, with practically no limits in pupae production.

ALGENEX appears to deliberately pinpoint the ‘pupae stage’ of metamorphosis when the caterpillar is transitioning from a worm into a butterfly to use the virus to hijack the body of the host. Why is the pupae stage so important?

Pupae are an inert stage of the insect evolution that takes in controlled  insectary conditions, while the insect undergoes metamorphosis. Cells at this evolutionary stage are encapsulated and isolated completely from the environment. However, the metabolism of these cells is extremely active, considering the dramatic changes that they are suffering transforming from a larva into a moth. In that sense, insects are unique. Some insect cells may produce 1.600 times more protein than the most productive mammalian cell. It means that the encapsulated cells may produce very efficiently our desired protein product. Pupae have relatively hard bodies and don’t move. It facilitates the automation of insect manipulation, storage and inoculation by specifically designed robots that Algenex has developed. It reduces manual operations, increases batch to batch consistency, reduces cost and boosts the scalability of production in this technology at levels never reached in insects. Pupae can be storage refrigerated for many days, allowing transportation or the programming of the production of new batches with high industrial flexibility.

How feasible is it to perform this sort of bug-tech on an industrial scale? Just how scalable is this process?

The use of robots at different stages of the process allows us to scale-up in unprecedented levels. As an example, with just 1 robotised inoculation machine of just one needle and a single standard incubator of the size of a home refrigerator, we are able to produce 2,400,000 doses per week of a purified dual vaccine for the prevention of RHDV in rabbits. We can further increase the scale by adding further needles to the robot or just putting more robots to work. As I mentioned before, the pupae supply is not an issue at all. Millions of our biocapsules are produced in a small facility in short periods of time.

How would you describe the true value proposition of your CrisBioÒ technology? What added benefit does it deliver up to the marketplace?

We describe CrisBio USP as follows:

• Simple and robust automated process
• Short development times for products
• Low investment, both CAPEX and OPEX
• High productivity, which can reach gram/litre of extract levels
• Production flexibility and linear & immediate scalability
• Products have the same quality of that obtained in insect cells cultured in bioreactors
• Proprietary and patent protected

How has the market received such a disruptive and novel approach to protein expression so far? How easy has it been to attract clients and investors?

As always, being the first is not easy, and it takes time for the industry to adopt new disruptive processes. In addition, most players have already invested in multimillion bioreactor facilities, which they need to justify. Having said this, we have been quite successful in securing interest which has translated into collaboration agreements and/ or licence agreements: We currently have five vaccines in our development pipeline, of which three have been partnered. Importantly, one of the vaccines has been recently partnered to an existing client, which is a proof of their satisfaction with the first project. We also have two agreements in place in the area of diagnostics. At the moment, we are in discussions with several companies interested in using our technology to produce non-vaccine products, including for human health. And we expect a breakthrough to come with the first EMA approval of a CrisBio-based vaccine, which we expect to come in the second half of 2020. Investor-wise, we secured a €5 million round in 2018, which was disbursed in two tranches. There are several well-known industry players who have participated in this round on a personal basis, further validating our technology and plans.

Where is the competition coming from? Are there any other firms out there trying to do the same thing or mimicking your activities? Will it be easy for others to copy your technology?

We are not aware of any serious competitor involved in the production of recombinant proteins using insect pupae. As I mention, we have IP on the process using pupae of our insect, and no other company has reached the level of sophistication of our production process, which include robots and single-use plastic devices for insect rearing, manipulation, storage and transportation. We are years ahead of any competitor that may appear in the future.

Companies in Asia using insects as biofactories are based in Japan and, for traditional reasons, they use Bombyx mori caterpillars. It requires a specific baculovirus vector to programme the cells, which is not used commonly by the industry or the research laboratories. In general, we consider these systems less efficient and more difficult to implement than our CrisBio technology. There are other companies with similar business models that use green plants or transgenic mammals as living biofactories.

What is the full potential and repercussions of utilising insects to manufacture proteins? How significant can this be as a new industry sub-sector? Do you expect it to really go mainstream? And, if so, when?

Rather than an industry sub-sector, we see our technology as a disruption of existing protein manufacturing processes that offers unlimited potential:

• As a solution to the capacity constraints faced in the first world for the production of protein-based therapies
• As a solution to create cost-effective therapies that can be made available in developing countries
• As a tool to efficiently produce a reduced number of doses to meet the needs of niche indications, including Orphan Drug products
• As a tool to rapidly respond to pandemics and disease outbreaks

The dossier for the first CrisBio-based vaccine has now been submitted to the EMA targeting centralised approval of this vaccine in 2020. This approval would imply the regulatory validation of our system, an important milestone towards developing products also for the human health segment.