Team:ULaVerne Collab/Human Practices
Insulin synthesis has been one of the major interests for many researchers and small-scale start-ups worldwide due to the world's diabetes demands that are not met by the high price of commercial insulin owned and distributed by a number of big companies. Many labs like ours are trying to change this fate of insulin by synthesizing insulin and making it open sourced so everyone can have better access to it. A major issue with that is choosing the appropriate chassis and designing DNA constructs that would allow for efficient and successful small-scale expression and purification of the insulin proteins. However, having reached out to a previous iGEM team that inspired our project idea, the Open Insulin team, and professors, we are able to overcome some of those challenges and are closer to reaching that goal. We also consulted them and experts in the medical field about the more viable application methods if our insulin were to be distributed to patients in the future.
Sydney Winsulin iGEM Team 2017
We know that insulin accessibility is a major issue, especially in the US, and we noticed that the Sydney 2017 iGEM team worked on synthesizing single chain insulin and did not seem to successfully get conclusive data from their construct designs, so that encouraged us to pursue that idea and learn more about the issue of insulin and diabetes.
Entrepreneur at Biofoundry - Alex Kelly
We reached out to Alex Kelly, one of the members and project designers of the Winsulin Sydney iGEM 2017 team. Currently he is a managing director of a lab called Biofoundry in Sydney, and still pursuing the same Open Insulin project.
Why did we approach him?
We wanted to discuss about the result of their project and figure out what we could improve on.
What did we learn?
We learned that when making the single-chain insulin (SCI) constructs they had modified or added the linker, AA substitutions, fusion protein tags, pI, while advising us to recheck and avoid the patent for the commercial Glargine and Lispro insulin, which helped point our construct design to the proper direction. He also pointed out to us that we do not need to make the insulin protein thermostable, as it is already stable during transportation. He is currently working on this project today, and encourages us to help him troubleshoot it too if we decide to do the project. He also introduced to us Open Insulin, a community aiming to manufacture Open Insulin, which his team collaborated with, and stressed to us about how important it is to make it open source so that big companies will not be able to monopolize the insulin industry like right now.
Physician - Dr. Francine Ratner Kaufman
We contacted Dr. Francine Ratner Kaufman who specializes in Endocrinology and has worked with diabetes for 30 years. She has written multiple books on diabetes and is currently working on creating insulin pumps for diabetic patients.
Why did we approach her?
We wanted to confirm how significant and applicable our insulin project goal and design would be in the future use from a physician perspective. Our goal is to make insulin cheaper by making it open-sourced, and available to anyone. For our design, we wanted to first increase the speed of insulin through the body, by making it single chain. We thought that if making insulin single chain in the lab is expected to work faster we could perhaps increase that speed by also improving our applied design. We also had a vision of administering insulin through patches embedded with microneedles or ingestion, which might increase the speed of insulin but mainly would alleviate the pain from frequent injections that patients of all age, especially children, receive each day. We also wanted to address the storage of insulin at places where there is a lack of access to refrigerators for long term storage, so we wanted to know what is the storage stability and shelf-life of insulin.
What did we learn?
We confirmed that patient’s biggest struggle besides the price is the frequent needle injections that they receive. However, she said that microneedle patches are topical and cannot pierce the skin any deeper into the subcutaneous layer where insulin should at least be in. Ingestion, on the other hand, would be degraded by the juices in the stomach. Regarding insulin stability and storage, she said that insulin is sensitive to extremely hot or cold temperatures, but at room temperature it can be stored up to 28 days. For long term storage, you can refrigerate it until its expiration date, but if it is already opened it would still be unusable within the 28 days. She also questioned if our insulin would still be in use in the next 20 years or so considering the medical and technological advancements of today, and how we would be able to fight against the pressure from big manufacturing companies if we were to distribute our insulins to the public.
Professor - Jennifer Tsui
Dr. Jennifer Tsui is a phD graduate from Stanford University and has a background in Neurobiology, Biochemistry and Molecular Biology. She teaches Molecular Biology at the University of La Verne and is our primary advisor.
Why did we approach her?
We were not sure what were the best amino acid substitution and linkers to choose for our project.
What did we learn?
She suggested that we could try building a library of single chain insulins and show a comparison of their efficiency, so the science community can utilize these data for future research.
Integrated Human Practices #1:
From our first conversation with Alex Kelly and our advisor’s help we were confident in designing our own single-chain insulin constructs for all the 3 most common types of insulin in the market, Insulin, Insulin Glargine, and Insulin Lispro. We started with 6 constructs in total initially, with different linkers, amino acid substitutions, and pI’s for those insulins. The amino acid substitution we decided on for the mutant long-lasting insulin (Glargine) is not in the patented list, and for our mutant fast-acting insulin (Lispro) we did not have to worry about the patent because it had been expired. We also added His and TEV tags for purification. Although we did not worry about insulin stability during transportation based on Alex’ advice, we worried about its storage at regions in the world with extremely hot temperature, which would benefit from making our protein thermostable. However, after knowing that it is also unusable in areas with extremely cold temperature, thermostability would not improve the quality insulin for these regions, so we decided to keep our project basic by excluding that factor. We also chose for our insulin to be injected after hearing the advice from Dr. Kaufman, as it would be indeed the fastest and most efficient way for insulin to be administered at the present. However, we still carried her questions with us and continued our journey to find the right people to advise us about those topics.
Professor - Jerome Garcia
Dr. Jerome Garcia is a phD graduate from the University of Southern California, with a focus on toxicology and pharmacology. He is a professor of Physiology and Immunology at the University of La Verne, and has done research on insulin.
Why did we approach him?
We wanted to review our insulin designs with him.
What did we learn?
He told us to make sure that our amino acid substitutions should not only be similar to the original amino acid, but should also avoid using amino acids that are listed in databases to disrupt the formation of disulfide bonds and the trypsin cleavage sites, which are important for the insulin protein structure. However, he told us that our insulin library project design might be too ambitious and time-consuming.
Open Insulin - Wet Lab Scientist - Yann Lala
Open Insulin is a non-profit organization of a small-scale start-up group of scientists, professionals, and volunteers from different fields working together, located at Counter Culture Labs in San Francisco, USA, and their goal is to manufacture and distribute an open-source insulin to patients with diabetes. Yann Lala is one of the scientists, who is in charge of the wet lab of Open Insulin. He has been working on synthesizing regular and long lasting insulin from yeast and, recently, E. coli.
Why did we approach them?
Due to Alex Kelly’s recommendation, we reached out to them, in hopes of asking about simple, effective but most cost-effective ways to synthesize insulin for small-scale startup labs like ours? besides using fusion protein tags for purification, like synthesizing each chain separately for the non single chain insulin. We also wanted to receive feedback for our single chain insulin constructs design (mostly regarding our placement of a His tag between proteins).
What did we learn?
We learned that Yann has been trying to express his insulin and long lasting insulin proteins using a yeast chassis, from which he struggled to get any expression at all. His project design is to synthesize insulin from preproinsulin by using three endopeptidases because his insulin is not single chain insulin like ours. In regard to our question about easier ways of synthesizing insulin, he suggested that for non-single-chain insulin synthesizing each chain separately is done in some manufacturers but very tedious for small-scale labs like ours. Since we are synthesizing single chain insulin it is easier to use protein tags for purification because if we were to try their method and find enzymes that can ultimately cleave our desired protein, there is a chance that the enzymes could recognize other similar sites in our entire sequence, which would be problematic. Regarding our placement of the His-tag being in between proteins instead of at the end terminus, he said from what he has read there is no conclusive data on that, but we can try to prove that it works. Based on our results later on, he also advised us later to add a flexible GGS linker to each side of the His tag to increase affinity to the nickel beads.
Data Scientist - Petr Ponomarenko
Why did we approach him?
We wanted to run a protein-receptor docking simulation of our single chain insulin with the 12AA linkers to the human receptor and predict the binding efficiency of our single chain insulin mutants. However, most of the programs we tried could not perform it. Therefore, we wanted an expert input on this issue and learn more about the ways experts do math modeling for drug molecules in the industry.
What did we learn?
Petr stresses that the type of drugs binding modeling we are trying to do is an extremely hard problem that a lot of big and small pharmaceutical companies struggle with. A lot of scripts, analysis, modeling and research has to be done for the binding simulation to work properly in our case. To make something relatively reliable and working we would need about 2,000 man work hours on the level of protein-protein 3d modeling expert with chemoinformatics experience of at least 5 years. A somewhat commercially useful results can be expected to appear around 100,000-200,000 man work hours. Basically, a team of 10-20 people with proper management can do it within 5 years for a handful of targets. The budget for such work starts around 20-40 million dollars including overhead and lab work. Thousands of different sequences’ thermodynamic data have to be used to train a model. But this is complex and requires a lot of data and expertise hence the high price. Nonetheless, we could still use simple docking programs to do it still, it just would not be as accurate and reliable. However, we can definitely get significant predicted data out of it such as interaction energy between the molecules and the side chain interactions.
Integrated Human Practices #2:
From the conversation with professor Jerome Garcia and advice from our advisor, we rechecked our amino acid that we input again to make sure they would not interfere with the original protein folding. We also decided to narrow our library to only proinsulin control and 3 single chain insulin mutants, which are our current constructs, due to time and resource limitations. Yann’s information helped confirmed our decision to use E. coli instead of yeast as our chassis, and to pursue our purification design using purification tags, which might be one of the least complex methods for a small-scale and lower-budget lab. Since our design includes certain protein tags for purification, it limits us to have a His tag in between bigger proteins, which we decided to keep that design add incorporate only the GGS linkers on each side of the His tag, in hopes of being able to report the efficiency of this novel placement of His tag being in between protein to the science and research community. Meanwhile, we were also attempting to perform insulin and receptor docking simulations, but we were not sure of the methods and significance of our model if we were to use it as supporting data for the manufacturing of our insulin mutants. Fortunately, Petr Ponomarenko cleared it up quite well that if we were to publish a reliable model we would need a lot of experts and time to train the model before the simulation can happen, and even pharmaceuticals are struggling to do it. Therefore, we ended up collaborating with the 2019 iGEM Moscow team, who ran a simple docking and interaction energy for us.
Open Insulin - Founder - Anthony Di Franco
Anthony Di Franco is the founder of Counter Culture Labs that hosts the Open Insulin organization since December 2015 and is the main person who manages all the workflow of the Open Insulin team.
Why did we approach him?
We wanted to understand the potential of this project into the future, in response Dr. Kaufman’s question for us.
What did we learn?
We learned that making insulin open-sourced, means making the protocol for insulin production open to everybody. During one of the weekly Zoom calls, a pharmacist who recently participated in the call, questioned him the question that Dr. Kaufman and we had about the significance of this project if future advancements might overpower insulin in the next several decades. As a response to that, Anthony brought up how those who cannot afford insulin cannot wait for who knows how long until the next insulin replacement is available to the public. Even if that were to happen soon, it would most likely be owned and perhaps patented by some companies again, which is the whole purpose of the Open Insulin community to get rid of that monopoly by making the protocols to producing insulin available to everyone. We also learned that the dream of the Open Insulin community is made possible by mainly the community support in terms of resources and finance. The Open Insulin project started by meeting a crowdfunding goal of $16,656 from the public, and ever since they have received funding from different parties, volunteers for different aspect of the organization, and resources and input from other labs, scientists, professionals, and the public.
Open Insulin - Social Scientist - Nicole
Nicole is one of the social scientists who is in charge of the legal aspect for the Open Insulin organization.
Why did we approach her?
We wanted to understand how the non-profit Open Insulin Organization are working towards achieving their goal against the pressure of the big companies and how they would protect their insulin without putting patents on it like the big companies do.
What did we learn?
We learned from her that for open-source production like open insulin, the whole purpose is to avoid putting a patent on it like the big companies. Instead they would have an ‘open source license’ or a GNU General Public License (GPL), which allows the protocol to be protected freely shared, used, and modified under certain terms and conditions. In order to obtain an open source license, we would have to go through the Open Source Initiative license review process and get approval by the Open Source Initiative. Meanwhile, in order to protect the protocol or product from others stealing it as their own, they would also have to put them under an intellectual property like a trademark or copyrights and not the patent. On the other hand, to protect their organization from other companies, they would have to have a lawyer by their side during the manufacturing process.
Entrepreneur at Biofoundry - Alex Kelly
We reached out to Alex Kelly again, one of the members and project designers of the Winsulin Sydney iGEM 2017 team. Currently he is a managing director of a lab called Biofoundry in Sydney, while still pursuing the same Open Insulin project.
Why did we approach him?
We wanted to understand the process of getting insulin analogue out in the market if we were to go beyond just making the protocol open to the public.
What did we learn?
We learned that it is an extremely lengthy, costly, and indeterminate process in order to get it to the market. Although biosimilars have to go through less clinical trials than normal drugs in order to get approved by the FDA, it would still cost more than 100 million dollars compared to the regular human insulin at around 15 million dollars. Then we would have to spend about 40% of our running expense to obtain a GMP (Good Manufacturing Practice) certification by the FDA, in order to assure that our manufacturing process meets their standards and regulations. Meanwhile, for the manufacture and power plant setup, we would have to spend from 5 - 500 million dollars depending on the scale of our manufacturing. After that we would have to spend on marketing and avoid a risk of price war.
Integrated Human Practices #3:
Talking with the Open Insulin members of different fields, helped us answer the question posed by Dr. Kaufman regarding the significance and potential of our project into the future and stay safe of bigger companies. Since the goal of Open Insulin is to make the protocol free to the public, even if newer drugs were to be released to the market, it would likely be patented and expensive again. Moreover, judging on the support and energy the open insulin project receives from the public and many professionals and sponsors, it is a project that does have a potential to move forward and successfully reach the goal if my labs like ours continue to help each other reach that goal. On the other hand, we learned that ways to protect our project during and after the manufacturing process is to be protected by a lawyer, and for protocols and products since our goal is to get rid of patents, we would still be able to protect them from identity theft and damages through an open source license and trademarks or copyrights. If we were to only successfully synthesize insulin and make it open source, these would be the main policies to go by. However, if we were to proceed to manufacturing and ultimately putting it out in the market by ourselves, as Alex explained, we would need a huge starting capital in order to go through the entire process of getting past the FDA. It would be more possible if a pharmaceutical or sponsor would sponsor us in that case, but again, going back to the purpose of Open Insulin, we would try to avoid dealing with parties that view insulin as a source of benefit and business. If we were to create a generic human insulin company, we and other labs that have access to our protocols would cap the price at an affordable rate since the protocols are open to the public anyway.
American Diabetes Association - Development Manager - Roman Garcia
American Diabetes Association (ADA) is a non-profit based in the US that helps educate the public about diabetes and help support research and cures for diabetes. Roman Garcia is a Development manager for the American Diabetes Association and was a representative at the ADA booth at the ADA Diabetes Walk.
Why did we approach him?
We wanted to share our project goal with them and learn what are different ways we can help support the community aside from the goal of our project into the future.
What did we learn?
Roman described about the many things that the organization and we can do for the public. One way is to form advocacy groups in different regions to educate the public and school about insulin, diabetes care, and most importantly care and prevention through exercising, which is a significant way the body can effectively absorb glucose through the GLUT 4 mechanism. Another big achievement that they have done was to advocate for insured diabetes patients to co-pay for their insulin at just $100 per month regardless of the amount they needed in the state of Colorado. He also mentioned that we should not worry too much about the big companies suing us because there is a lack of policies surrounding insulin and pricing at the moment. How insulin prescription are priced so high are still not clear to even state attorneys, who are investigating into the process of pricing insulin and reviewing legislations. However, the ever-increasing price is likely to be attributed to most of the stakeholders from the manufacture to the pharmacies or distributors. He believes that first reevaluating policies to control the price of insulin at different stages from manufacturing to patient distribution is the first step to controlling the price of insulin. Meanwhile, he agrees that our open source insulin would definitely bring competition to the big companies who keeps increasing the price of insulin as well, while we are waiting for better policies in the next many years to come.
ADA Diabetes Walk - Staffs and Participants
Why did we approach them?
We wanted to ask people who are to blame for the high price of insulin. Most importantly, we wanted to learn about people's experiences or thoughts at the Diabetes Walk, which is an important gathering that celebrates everyone who is living with Diabetes.
What did we learn?
We chatted with many people, who were young, old, work for the stakeholders, has had family members living with diabetes, is a diabetic patient, or just wanted to support the diabetes community. When we asked what they think contribute to the high price of insulin, most of them mention the insurance companies, pharmaceuticals, and manufacturers. A pharmacist said he believe it is the pharmaceuticals mainly, but most likely every stakeholder as well. Another man that works for one of the big pharmaceuticals, said that he believes it is less of the pharmacies problem but more of the pharmaceutical benefit managers. We also talked to an ADA staff member has lived with diabetes for 46 years. She shared how she is completely reliant on insulin for her whole life and has a tube on her lower stomach for regular insulin administration. She complained about how the price of insulin ridiculously increased, from which she has to pay for more than $200 for every two months, as she had to switched to a newer national health insurance program, and believe that our project will help change that. She also believes that stakeholders are responsible for the high price of insulin because she said her insurance charged her for the insulin every two months while she receives her glucose reader and insulin injector for free when they are supposed to be more expensive. Also, even though insurance companies do cover usually 82%, patients would still have co-pay within the range of $20 to more than $60 depending on the type of insulin and brand. Another man who works for an engineering company said that this issue is complicated to pinpoint because there are a lot of contributing factors like the benefits that stakeholders get. However, he believes that the government can help alleviate this confusion with the increase of the price of insulin by investing more in medical research to develop insulin alternatives, as a way of tackling the root of the problem.
Integrated Human Practices #4:
From our conversation with Roman, we learned about the mission and support that our local American Diabetes Association give to the diabetic community, which we can spread the message about them to other people we know that need that support. Meanwhile, we ourselves and other people that are interested in joining that campaign, can also start our own advocacy groups and volunteer for the ADA to spread the message and educate others about the disease, care, and prevention. We also learned that, although our goal is to create open source insulin as a way to lower the market price tag, there are other ways that we can achieve that goal too through policy advocacy. If we, the ADA, and more people start to advocate in each state to cap the price of insulin for patients like how it was done in Colorado lately, that might help increase the rate of affordability within the country for the time being since there is still no clear policies about the regulation of the complex web of insulin pricing. Moreover, an important information that I learned from talking to the public coming different backgrounds is that nobody knows which stakeholder is responsible for the high price of insulin exactly. The fact that the government still has not been able to manage the policies for pricing insulin is shown within the confusion and struggle that the public express. Fortunately, more researchers are doing research and digging into the roots of insulin journey and pricing from the point it is manufacture to distribution to different countries, to taxes that the country adds, to insurance companies, pharmaceuticals, and whatnot until the drug reaches the patient (Beran, 2016). Therefore not until the policies are sorted out or the open insulin project succeeds, the hope of making insulin affordable and accessible to patients looks dim.
Reference:
Beran, David, et al. “Constraints and Challenges in Access to Insulin: a Global Perspective.” The Lancet Diabetes & Endocrinology, vol. 4, no. 3, 2016, pp. 275–285., doi:10.1016/s2213-8587(15)00521-5.