Our team went through the proposal and rejection of several different ideas for this year’s project. After considering aspects such as feasibility, cost, and most importantly, impact, we chose to pursue the issue of heparin production. 

Many of us were surprised to learn that heparin, the substance that lines IV tubes and is the most common anticoagulant used in the medical field, is derived from pig intestines and primarily sourced from China. Not only does this pose significant ethical concerns, but procuring such a crucial pharmaceutical product from a foreign country introduces a degree of instability and unreliability. Given that most medical patients are unaware of the risks associated with using heparin, and that alternative anticoagulants are often too costly and inefficient for widespread use, our team chose to investigate a way to biologically manufacture this essential anticoagulant.
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Around 12 million hospitalized patients in the US receive heparin during a variety of medical procedures, including cardiovascular and orthopedic surgery and invasive procedures, acute coronary syndromes, peripheral occlusive disease, dialysis, and extracorporeal circulation [2]. Heparin is also used in intravenous catheters to prevent blood clots from forming when it’s first inserted as well as every time blood is drawn from the patient [3]. The anticoagulant is also administered long-term for certain conditions, including venous thromboembolism, atrial fibrillation, and certain cancers. To treat these illnesses, a predesignated dosage of heparin is administered regularly for a few months. 

Warfarin is another common anticoagulant used in the field. However, warfarin takes up to around 5 days to take effect and works by reducing the amount of vitamin K in the body, requiring the patient to consume vitamin supplements. On the other hand, the effects of heparin can be quickly started and reversed using protamine, allowing a level of control should unexpected bleeding or clotting occur. In addition, heparin does not cross the placenta of pregnant women while warfarin does, which puts the fetus at significant risk. Other anticoagulants, such as fondaparinux and enoxaparin, are limiting for  rural hospitals due to their high costs compared to the low price of heparin [4] [5].
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There are two main types of the anticoagulant: unfractionated (UFH) and low molecular weight heparin (LMWH). LMWH is produced by further modifying and purifying UFH. The heparin extraction process begins by immersing the cleaned intestines in a brine solution, followed by the scraping of the mucosa. Then, proteases and chemical treatment procedures are used to collect the heparin from mast cells and heparin is isolated from other small molecules using soluble quaternary ammonium cations. Other impurities, including other glycosaminoglycans (GAGs), are precipitated out of the mixture using organic solvents [6]. This process produces crude heparin, or unfractionated heparin, and has a molecular weight that ranges between 3,000-30,000 Da and averages around 12,000–16,000 Da. 

To produce LMWH, UFH is subjected to controlled depolymerization through enzymes and chemicals which cuts the UFH heparin polysaccharides into smaller, stable sizes. The molecular weight of LMWH ranges from 1,000-10,000 Da and averages around 4,000-5,000 Da [7]. LMWH has a longer half-life than UFH, thus requiring fewer doses per day. The relative uniformity of the heparin polysaccharides in LMWH results in more predictable anticoagulant activity levels and therefore doesn’t depend as much on lab monitoring. Furthermore, LMWH molecules are less likely to bind PF4 molecules, lowering the risk of developing heparin-induced thrombocytopenia (HIT) [8]. 

However, LMWH is not a completely pure substance, has limited access to rural hospitals due to its high cost, and is animal-derived. By biomanufacturing heparin in E. coli, we hope to further increase the uniformity of the heparin molecules, reduce risk of HIT, and resolve any ethical concerns involving porcine-derived products.
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Heparin-induced thrombocytopenia is the most common side effect seen in patients who have been administered heparin. HIT develops in two distinct forms, type I and type II. Type I HIT develops in around 10% of patients and is characterized by the development of temporary, mild thrombocytopenia, a condition that describes a patient with low platelet levels in their blood. This response is non-immunologic and is due to the clumping interaction between heparin and the surrounding platelets. The response develops within 24-72 hours of heparin administration and returns to normal within 4 days after the heparin is withdrawn [1]. 
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On the other hand, type II HIT occurs in up to 5% of patients who have been exposed to heparin products and involves an immunologic, antibody-mediated response. Type II HIT may lead to deep venous thrombosis, pulmonary embolism, shin necrosis, myocardial infarction, or venous gangrene, and has a mortality rate of up to 30%. The response begins when heparin initially binds to the platelet-forming factor 4 (PF4) molecule released from the alpha granules of platelets upon activation. In some patients, this may lead to the formation of a class IgG antibody that binds to the FC receptor of the heparin-bound platelets. This activates the platelets, causing them to release pro-thrombotic substances, which, in turn, activates more platelets and releases more PF4 molecules. The positive feedback loop created by this cycle creates a sensitive, hypercoagulable state and can easily lead to venous or arterial thrombosis [9]. Lepirudin is a direct thrombin inhibitor and is considered the preferred treatment for type II HIT, reducing the risk of thromboembolic complications by 92.9%. However, anti-lepirudin antibodies are produced in up to 56% of recipients, and may lengthen the drug's half-life by interfering with renal clearance. Additionally, anaphylaxis occurs in 0.16% of patients after lepirudin re-exposure, putting patients at risk when repeatedly utilizing this treatment [10]. 9,600-48,000 people per year develop HIT with thrombocytopenia, and is a major health effect that arises from the current form of heparin.
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The first major swine epidemic that shook the heparin industry occurred in 2008 and resulted from the rapid spread of the viral disease porcine reproductive and respiratory syndrome (PRRS) in China, causing drastic inflation of pork-related products. This price rise led manufacturers to add over‐sulfated chondroitin sulfate (OSCS), a type of chondroitin sulfate sourced from mammal or shark cartilages, to mimic the anticoagulant properties of heparin. However, this resulted in hypotension and allergic reactions, ultimately bringing about more than 80 deaths and almost 800 injuries. A decade later, the African Swine Flu (ASF) caused by the asfivirus (Family Asfaviridae) began wiping out hundreds of thousands of pigs in China in a matter of months. The small, enclosed farmhouses further exacerbated the spread of the disease and majorly inflated the prices of porcine products once again. Many drug agencies agreed to create bovine-derived heparin to avoid reliance on a single exporter of heparin. However, heparin in the industry today is still majorly sourced from these pigs [11].

These instances demonstrate the clear instability of using a medical product sourced from animals, which can be avoided by utilizing bacteria to produce the heparin.
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[1] Ahmed I, Majeed A, Powell R. Heparin induced thrombocytopenia: diagnosis and management update. Postgrad Med J. 2007 Sep;83(983):575-82. doi: 10.1136/pgmj.2007.059188. PMID: 17823223; PMCID: PMC2600013.
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[2] Chong BH. Heparin-induced thrombocytopenia. J Thromb Haemost. 2003 Jul;1(7):1471-8. doi: 10.1046/j.1538-7836.2003.00270.x. PMID: 12871282.
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[3]“Heparin Injection: MedlinePlus Drug Information.” Medlineplus.gov, 2017, medlineplus.gov/druginfo/meds/a682826.html. Accessed 18 Aug. 2024.
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[4] Pineo G, Lin J, Stern L, Subrahmanian T, Annemans L. Economic impact of enoxaparin versus unfractionated heparin for venous thromboembolism prophylaxis in patients with acute ischemic stroke: a hospital perspective of the PREVAIL trial. J Hosp Med. 2012 Mar;7(3):176-82. doi: 10.1002/jhm.968. Epub 2011 Nov 4. PMID: 22058011.
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[5] Robert Chad Hakim, and Kristianne Hannemann. “Fondaparinux (Arixtra): Uses, Dosage, Side Effects & More.” GoodRx, 17 Aug. 2021, www.goodrx.com/fondaparinux/what-is. Accessed 18 Aug. 2024.
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[6] van der Meer JY, Kellenbach E, van den Bos LJ. From Farm to Pharma: An Overview of Industrial Heparin Manufacturing Methods. Molecules. 2017 Jun 21;22(6):1025. doi: 10.3390/molecules22061025. PMID: 28635655; PMCID: PMC6152658.
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[7] Hirsh, Jack. “Low-Molecular-Weight Heparin.” Circulation, vol. 98, no. 15, 13 Oct. 1998, pp. 1575–1582, https://doi.org/10.1161/01.cir.98.15.1575. Accessed 16 June 2020. 
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[8] Lawrence, Peter F. “Chapter 78 - Pharmacologic Adjuncts to Endovascular Procedures.” ScienceDirect, W.B. Saunders, 1 Jan. 2011, www.sciencedirect.com/science/article/abs/pii/B9781416062080100783.
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[9] Nicolas, Diala, et al. “Heparin-Induced Thrombocytopenia.” PubMed, StatPearls Publishing, 2023, www.ncbi.nlm.nih.gov/books/NBK482330/. 
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[10] Adam C. Cuker, Chapter 100 - Heparin-Induced Thrombocytopenia, Editor(s): Beth H. Shaz, Christopher D. Hillyer, Mikhail Roshal, Charles S. Abrams, Transfusion Medicine and Hemostasis (Second Edition), Elsevier, 2013, Pages 651-662, ISBN 9780123971647, https://doi.org/10.1016/B978-0-12-397164-7.00100-2. 
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[11] Eduardo Vilanova, Ana M.F. Tovar, Paulo A.S. Mourão, Imminent risk of a global shortage of heparin caused by the African Swine Fever afflicting the Chinese pig herd, Journal of Thrombosis and Haemostasis, Volume 17, Issue 2, 2019, Pages 254-256, ISSN 1538-7836, https://doi.org/10.1111/jth.14372.

[12] Scientific Image and Illustration Software (2017) BioRender. Available at: https://www.biorender.com/ (Accessed: 25 August 2024).

[13] A man disinfects a pig farm in Guangan, Sichuan province, China August 27, 2019. (2019, August 26). Reuters. https://www.reuters.com/article/business/healthcare-pharmaceuticals/chinas-sichuan-province-to-remove-restrictions-on-pig-farming-idUSKCN1VH09V/
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