Overview
Methylxanthines, a molecule naturally occurring in caffeine and its derivatives, are prized in the pharmaceutical industry for their use as a stimulant and as a bronchodilating agent [1]. They are also a crucial component of novel myopia treatments, the focus of our project [2]. Current production methods utilize an inefficient chemical synthesis that is costly both in terms of time and funds. Our goal was to use enzymes to do the “heavy lifting”, developing a more efficient and economical process to synthesize methylxanthines . In doing so, we hope to advance the development of myopia treatments by making a critical component for said treatments more accessible, affordable, and easier to produce.
In order to do this, we needed to develop an environment that would be conducive and efficient for this biochemical procedure. Initial considerations centered around batch process reactors that iGEM Cornell has worked extensively with in the past. Despite prior successes, a batch bioreactor lacked the necessary properties for the enzymatic synthesis that this project required. To this end, we opted to instead design a fixed-bed bioreactor in which our selected enzyme would be immobilized in alginate beads and those beads then immobilized within the bioreactor. One benefit of this setup is that the reactor is cell-free, eliminating biohazard risks that may be present in other reactor designs. Another is that the reactor itself is easy to maintain, as the beads are not free-floating in the medium, as is the case in a batch reactor.
Enzyme Immobilization
Part I: Bead Creation
Simply flowing enzyme with reactant through the solution was the most basic, preliminary model that we considered. This model would ensure that all the reactants could be converted into products given enough time, measured through Kcat and Km and using enzyme kinetics. However, the drawbacks of this approach would be that the enzyme would remain in solution and flow out of the reactor as well, leaving another compound to separate as well as a need to replenish the supply of enzyme per iteration. Thus, we moved towards immobilizing the enzyme in the system to increase efficiency.
During initial brainstorming processes, several methods for enzyme immobilization were considered. Encapsulation ideas included immobilizing enzymes within a porous membrane in beads (via sodium alginate, calcium alginate-clay, silica, or chitosan beads), linking them to a matrix of hydrogen bonded organic frameworks, or capturing the enzymes within a system of magnetic nanoparticles and an organic framework linked to magnetic material.
Magnetic nanoparticles proved too expensive, and an organic framework throughout the reactor was not feasible with the time and resources available to us. As a result, we decided to proceed with encapsulation of the enzymes within a porous membrane.
The materials required to create sodium alginate beads, sodium alginate and calcium chloride, were already readily available to us, and were simple to work with. Furthermore, Dr. Phillip Milner, a Professor of Chemistry and Chemical Biology at Cornell university, recommended sodium alginate to encapsulate enzymes, stating that it would be the cheapest and simplest solution, offering the least complicated scale-up processes. Thus, we moved forward with the creation of sodium alginate beads as an encapsulation method.
Before encapsulating any enzymes, several experiments needed to be performed to determine the ideal calcium chloride and sodium alginate concentrations to form beads that would not break under pressure, maintained uniform concentrations of enzyme throughout the bead, resulted in uniform bead shape, and allowed sufficient mass transfer through the porous membrane.
The calcium chloride concentration experiments were performed using a constant sodium alginate concentration of 3% weight by volume. In these experiments, no noticeable differences between bead properties were observed, and it was determined that calcium chloride concentration had no effect on bead property.
Sodium alginate concentrations were tested using a constant 1M calcium chloride concentration. During these experiments, it was observed that beads at a lower concentration were smaller and more uniform in size and shape, but also prone to breaking under pressure. The beads formed with higher concentrations of sodium alginate, on the other hand, were less uniform in size and shape but much less prone to deformation and breakage under pressure.
The tests conducted to verify optimal sodium alginate concentration can be seen in Figure 1 and 2; the uppermost row demonstrates beads that were split with a sharp tool, the second demonstrates untouched beads, and the lower row demonstrates beads on which flat pressure was applied (notice how 1.5% and 2% weight by volume split under this pressure).
When the sodium alginate concentration exceeded 2.5% weight by volume, the beads demonstrated an incredibly heightened amount of “tailing”, the property we used to describe the issue where beads form a teardrop shape rather than a perfect sphere. The sodium alginate itself was also much more difficult to handle and drop into the calcium chloride solution a single drop at a time. Furthermore, an increase in sodium alginate concentration increased the durability of the beads. Beads under 2.5% weight by volume could be split open under pressure, whereas 2.5% weight by volume and above could only be split with a sharp object.
Through all of these experiments, it was concluded that the sodium alginate beads necessary for encapsulating our enzymes should be formed with a 1M calcium chloride solution and a 2.5% weight by volume sodium alginate solution.
Part II: Encapsulation Automation Creation
After determining the ideal concentrations for the calcium chloride and sodium alginate solutions, it became evident that we needed to create an improved production mechanism that could both speed up and uniformize the bead-making process. Creating beads by hand was tiring and time-intensive, as it required very precise pressure on the syringe, and created very inconsistent beads, as any changes in pressure resulted in beads with air bubbles, beads with incredibly long tails, or large sodium alginate blobs that no longer classified as beads for our purposes.
The mechanism for the automatic bead maker is rather simple; using the same syringe required to make the beads by hand, it was set up inside a syringe infusion pump. By keeping the eight inch needle on the end of the syringe, and placing a container of calcium chloride solution several inches underneath the syringe tips, the beads leave the syringe relatively uniform and have time as they fall to regain a spherical shape.
In setting the infusion rate of the sodium alginate to 8 milliliters per minute, we also created a very uniform bead size. This is a result of the surface tension and viscosity of the sodium alginate; because the solution is uniform, the amount of solution that is pushed out of the syringe before it falls off the tip and into the calcium chloride is almost exactly the same every single time a bead is made.
Utilizing this process, we were able to make almost 70 beads per minute. This is incredibly efficient, as only around 50 beads are needed for a single batch in the bioreactor.
Reactor Design
The first step we took with regards to designing our fixed-bed bioreactor was designing the vessel that we would be employing. Firstly, a cylindrical geometry was selected for CAD model adaptability and compatibility with motor-driven flow to move the reactants. Following extensive material research, we decided to use PVC piping for its low reactivity, ease of procurement, and cost efficiency. A CAD model was created and then 3D printed as a physical structure to immobilize the alginate beads in place. The benefits of this immobilization are discussed in more detail below.
Turning back to the structure, we designed the framework to be as modular as possible to assist in another one of our major goals headed into this project: scalability. Because the only custom part required for the reactor itself is the immobilization structure, scaling up the synthesis is a simple matter of adjusting and printing additional frameworks for every reactor desired to be running in parallel or in series. For testing purposes, a 10mL syringe was used as the initial vessel due to their ease of procurement and simple geometry as well as compatibility with tubing and motor specifications. Additionally, the syringes that were used were made of propylene (PP), a material similar to PVC but used for more medical applications than PVC due to less toxic manufacturing procedures. It shares the same economic feasibility of PVC in addition to its non-reactiveness.
In parallel with the framework design, various parameters of flow were also tested to determine their optimal values. These parameters include motor voltage, incline, length of tube, and the cross-sectional area of the tube. These exploratory tests were conducted using a mock-up built out of a 1.5’’ x 2’ PVC pipe with plastic hobby beads to simulate our alginate beads. The large PVC pipe was chosen over the 10mL syringe for future scalability considerations. These experiments also allowed the exploration of the concept of residence time as pertaining to our final product. On the recommendation of Professor Christopher Alabi, we searched existing texts for mathematical ways to model residence time. However, because our design consisted only of our mockup at the time, these methods proved infeasible.
Thus, we worked to develop a new means of measuring residence time to meet the demands of our unique setup. The way we sought to do this was by modulating various parameters of our mockup, such as the power provided to the pump or the amount of beads in the PVC tube. However, the issue encountered was similar to the previous literature: the mockup didn’t allow for the complexity necessary to determine an accurate mathematical relationship between the flow parameters and the residence time. Thus, residence time was not a reliable measurement that we could use for further design considerations.
Once the design for the reactor itself was fairly solidified, we then shifted focus to immobilizing the alginate beads. To this end, we designed and 3D-printed immobilization prototypes that would allow us to hold alginate beads containing the enzyme in place mechanically. The immobilization methods we developed sought to use various structures to hold the alginate beads in place without the need for additional chemical reactions, which may have led to complications with the conversion of methylxanthines. These reactions are also, as mentioned earlier, very expensive.
One of the experts we interviewed, Cornell Professor Sijin Li in the Smith School of Chemical and Biomolecular Engineering, recommended 3D printing an immobilization framework in order to customize the immobilization configuration desired for the beads. With this advice, we began to create CAD models of potential immobilization methods. The prototype was edited to fit within the physical constraints of our vessel. We narrowed down to one prototype (as seen in Figure 3) due to its ease of printing and the design’s conduciveness to the beads settling individually up the spiral structure once flowed through.
The final product had our enzymes encapsulated in alginate beads, which were immobilized using the 3D-printed scaffold seen in Figure 3. These scaffolds were placed in 10mL syringes connected to an individual flow circuit through which substrates were flown in and methylxanthine derivatives were flown out, propelled by a simple fluid pump assembly. This design is easy to replicate and modular, the only specialized part being the scaffold and beads, which can be easily replaced should defects occur.
Future Steps
The success of our current design, specifically the ability of our enzyme beads to interact with reactants within our bioreactor, is tested through a simple experiment that determines whether reactants can enter and exit the beads. This is achieved by flowing dyed water through the reactor, and comparing the color of the beads within the reactor to the color of beads that had only ever been exposed to transparent calcium chloride. If the beads have become a closer color to that of the dyed water, we can confirm that the water is able to enter and exit the pores in the beads and given enough time, sufficient mass transfer can occur for an enzymatic reaction..
A natural extension of this work is scaling the system up for industrial application and manufacture. One way we propose doing this is to create a parallel system by which one inflow feeds into several, identical tubes whose outflow is combined. Of course, the flow rates would need to be calculated with respect to residence time and other dynamic factors to ensure proper function. However, when assembled correctly, this system’s size is limited only by the maximum flow rate of the fluid motor pump. Additionally, the use of identical tubes lends modularity: if one tube underperforms or for whatever reason fails, it can be easily replaced by another, identical tube. In short, this hypothetical scale-up could be highly modular, economic, and able to be easily scaled up (or down) depending on desired throughput.
However, this setup requires robust and thoroughly-tested parts. To this end, we would, in the future, seek user testing. We would ideally do this by providing our product to various entities in fields related to this project, such as the pharmaceutical industry or laboratories with specific needs of difficult to synthesize biomolecules. Then, we would collect feedback from said entities, improve the project, and repeat the process. Furthermore, user input will allow us to further optimize our parameters, such as improving our immobilization prototype or optimizing the ideal residence time per run. Additional optimization would include testing different motors, tubing materials or styles, and various flow parameters like flow velocity and volume.
References
[1] Gottwalt, B., & Tadi, P. (2023). Methylxanthines. In StatPearls. StatPearls Publishing. http://www.ncbi.nlm.nih.gov/books/NBK559165/
[2] Trier, K., Munk Ribel-Madsen, S., Cui, D., & Brøgger Christensen, S. (2008). Systemic 7-methylxanthine in retarding axial eye growth and myopia progression: a 36-month pilot study. Journal of ocular biology, diseases, and informatics, 1(2-4), 85–93. https://doi.org/10.1007/s12177-008-9013-3