Dr Finola Cliffe, Anne O’Sullivan and Prof Mark Davies outline the development of Enigma, Hooke Bio’s microfluidic high-throughput drug analysis platform, and its benefits for the pharma industry
Chem

Hooke Bio is an emerging biotech company focused on the development of high throughput and high content testing platforms for faster and more effective drug screening.

There is an important and unmet need for new medicines that can be mediated by the use of ultra-high-throughput microfluidic screening. In drug discovery, pharmaceutical companies screen hundreds of thousands of possible drug candidates against disease models.

However, some 95% of new drugs fail during development, with many failures attributed to efficacy and safety issues as the industry currently does not have the capability to screen every possible drug candidate, concentration or vary the drug efficacy safety tests due to sheer numbers.

In addition, the average cost to launch for a successful new drug can often exceed $2.5 billion and preclinical drug development is responsible for about 30% of the cost. High-throughput screening (HTS) is required to screening vast numbers of drug and drug combinations, as their effects on cells are difficult to predict. As a result, the testing of such drugs becomes an engineering issue.

HTS assays are used for screening huge drug-candidate libraries, including combinatorial chemistry, genomics, protein, peptide libraries and natural novel drug compounds. It has been estimated that the actual number of these possible drug candidates could be 1 novemdecillion (that’s 1 with 60 zeros).

Current approaches to HTS employ microplates with densities ranging from 96-1536 wells/plate, and reaction volumes ranging from 1mL to 1µL. The primary concerns with existing technology are the reagent costs and limited throughput.

The pharmaceutical industries have replaced human scientists that traditionally manually filled wellplates with robotic arms/technologies. This is essentially an example of batch processing and is widespread across the industry. Droplet-based microfluidics holds enormous potential for transforming the way that pharmaceutical companies screen for new drugs and drug combinations.

Prof Mark Davies saw the opportunity in this and founded Hooke Bio Ltd in March 2014, bringing together a multidisciplinary team of engineers and scientists. He was confident that an engineering solution could be developed to address the lack within the pharmaceutical industry. Within three years, the Enigma platform was prototyped and developed to demonstration readiness. This speedy innovation was aided by his prior experience as co-founder of Stokes Bio, which sold to Life Technologies in 2010 for US$44 million.

How tech works and who benefits


The concentric rings and parallel lines of the Enigma platform which are used to generate and transfer multiple droplets at the microscale

The Enigma device is an ultra-high throughput, cell-screening platform that utilises multiple parallel lines to test combinations of cells and drugs using droplet microfluidic technology. The unexploited power of microfluidics lies in the counter-intuitive behaviour of fluids on the microscale.

The Hooke Bio design allows picking of aqueous droplets on the nanolitre scale from reservoir wells. Each well contains a different component, e.g. cells, drugs, reagents and droplets, which can be comprised of multiple different wells depending on the experiment being conducted. The tiny droplets are separated from each other by an immiscible carrier fluid, allowing each droplet to serve as an isolated reaction container.

Enigma is a fully automated system requiring minimal human interaction. The general operation sequence involves loading the cells and drugs into the test reservoir wells, picking various droplet combinations, incubating the droplets containing cells and drugs/drug combinations, and subsequently analysing the incubated droplets using the detection method of choice.

Microdroplet-based HTS therefore has the potential to reduce assay volumes, increase throughput and eliminate the need for liquid handling robots, all of which result in significant savings. Costings analysis shows that the throughput for the initial Enigma platform will be 16x that of current systems, capex for the platform will be 3x-4x less and the consumables costs will be 35x less than existing systems.

The name Enigma is inspired by the wartime rotor-cipher coding machine used during World War II. As Enigma is designed as an annular device, it is particularly suited to screening for suitable drug combinations against various cell lines. Enigma is particularly beneficial to smaller drug-screening companies or academic research groups, as it is a low-cost instrument that would allow them to enter the screening market.

Many competitive technologies have capex costs of more than a million euro, which is a substantial barrier to entry. The main primary user is the pharmaceutical industry, which accounts for 48% of the high throughput screening market. This is projected to be worth $12 billion by 2019.

Improved biomimics and testing


Hooke Bio’s cell tests can be undertaken in 3D cell culture, which is a better biomimic of normal physiological processes than current traditional cell culture methods. Improved biomimics will result in fewer false positives being brought forward for testing in later more expensive and time-consuming testing rounds.

The ultra-high throughput interactive design of the Enigma platform means that personalised medicine and drug screening can become a reality. For example, a patient may have a sample of their cancer cells cultured in the platform and, subsequently, all possible combinations of relevant cancer drugs could be screened against that patient’s cancer to identify the optimum treatment regimen.

This is known as personalised combination screening and would it remove the element of uncertainty that surrounds chemotherapy (reduced likelihood of drug resistance by cancer cells emerging). Using multicellular cultures to form more personalised representative disease models such as spheroids and organoids would also aid this process.

Pharmaceutical companies are employing combination screening and drug repurposing to treat a wide range of diseases including, but not limited to, cancer and heart disease mainly using ultra-high-throughput screening.

Another example of the possible implementation of our platform is in the area of personalised medicine using a patient’s own reprogrammed cells, specifically induced pluripotent stem cells (iPSCs).

These cells can be differentiated into the cells for which drug treatment is required (essentially clones of a patient’s own cells) and such a personalised pathway mitigates efficacy and safety issues with drug regimens. This is particularly suited for genetic disorders or diseases such as type I diabetes and would allow faster, cheaper drug development.

Frequently, the best way to enter an established market is on the back of a disruptive technology. The innovations around iPSCs and CRISPR (gene editing) technologies would be optimal to allow Hooke Bio to successfully enter the market. Due to the ability of Enigma to screen vast numbers of compounds and by possibly implementing personalised medicine, an additional benefit is that animal testing may become obsolete.

Overcoming hiccups along the way


Hooke Bio’s droplets are aqueous droplets surrounded by an oil layer to allow flow. Although this format is ideal for short-term biochemical and cell culture tests up to 48 hours, beyond this time period the nutrient supply to the cells becomes fatally reduced.

To rectify this and to allow long-term cell culture (>48 hours) on Enigma, we have developed giant unilamellar vesicles (GUVs). These are water/oil/water emulsions that allow diffusion of nutrients across the oil membrane and removal of waste products. This would allow us to develop specific disease models within the GUVs for indications that require much longer cell tests such as type 1 diabetes.

We have also developed a third microcarrier called a cavity and, at present, Hooke Bio has three microcarrier formats that can be selected depending on the cell test.

The design of Enigma requires macro engineering at the microscale. The precision required to repeatedly produce droplets across multiple parallel lines may be compared to the design of a space telescope or that of a mechanical watch.

The inner workings of Enigma house a series of gears, pulleys and bearings that drive the rotor cipher machine through large distances to generate micro-scale droplets. Great care must be taken to remove both internal vibrations from the gearing system, which provides lateral and torsional vibrations, but also from external sources, such as people passing by.

Careful consideration was given to designing a robust system that was capable of traversing through rotations while maintaining the accuracy and precision required to complete meaningful experiments. Reservoir wells may also be individually thermally controlled to adapt to the biological and fluidic requirements of the compounds to be tested.

The pharmaceutical industry is very conservative and it will be a challenge to overcome its reliance on wellplates and robotic arms. However, the advent of new technologies such as CRISPR and iPSC may provide Hooke Bio with the opportunity to break such traditional thinking. That is the current Hooke Bio challenge.

Achievements and future plans


Hooke Bio Ltd was spun out from the University of Limerick in June 2017 and, to date, has secured €1.3 million in funding from private and exchequer sources. This investment will fund the development of the company two-to-three years into the future. Hooke Bio was also recently named ‘one to watch’ at Enterprise Irelands Big Ideas showcase in June 2017.

Currently Hooke Bio has six patents securing the technologies surrounding Enigma with more planned for our ongoing research activities. We are also in the process of recruiting engineers with plans in place for recruiting of additional staff over the next few months.

In terms of market entry, Hooke Bio will first target pharma industry to develop relationships with large pharma organisations. Such partnerships will have milestone-driven payments and give the partner access to Enigma. Partnership revenue will be sufficient to bring the Enigma platform through CE marking and to market readiness.

A sales team would be put in place to coincide with the market launch of the device. Sales of the instrument would focus on the platform itself and technology upgrades to suit the needs of individual users, consumables sales and maintenance contracts. We hope that Hooke Bio will make the world a safer place in which to live.

Authors:
Dr Finola Cliffe is chief operations officer with Hooke Bio Ltd. Anne O’Sullivan is a PhD student in the University of Limerick. Prof Mark Davies is professor of engineering science in the University of Limerick

http://www.engineersjournal.ie/wp-content/uploads/2017/10/Enigma-platform.pnghttp://www.engineersjournal.ie/wp-content/uploads/2017/10/Enigma-platform-300x300.pngMary Anne CarriganChembiopharma,biotechnology,industry,pharma,research
Hooke Bio is an emerging biotech company focused on the development of high throughput and high content testing platforms for faster and more effective drug screening. There is an important and unmet need for new medicines that can be mediated by the use of ultra-high-throughput microfluidic screening. In drug discovery,...