Paulo MourÃ£o is professor at Federal University of Rio de Janeiro.

Paulo Mourão

Heparin regulates hemostasis at various points of the coagulation cascade mainly through its interaction with antithrombin and heparin cofactor II. Because of these properties, heparin is a life-saving anticoagulant drug used in renal dialysis, cardiac surgery, and treatment for deep vein thrombosis. The drug also binds to platelets, inhibiting platelet function and contributing to the hemorrhagic effects of heparin. Bovine heparin, first approved in 1939, was widely used in the United States for more than 50 years (see 

). Like all drugs, heparin can cause adverse effects, but overall, bovine heparin products were found to be safe and effective during that period.


	
	In the late 1980s, bovine spongiform encephalopathy (BSE, or “mad cow disease”) was reported first in the United Kingdom and later in several other countries, raising concerns about the use of bovine-sourced heparin products in humans. Because of these concerns, manufacturers of bovine heparin products voluntarily withdrew them from the US market in the 1990s. Since then, heparin products approved for use in the US and Europe have been sourced solely from pigs, with approximately 60% of the supply of the drug coming from China. 

 illustrates steps involved in manufacturing heparin from porcine intestinal mucosa and potential impurities that are inactivated and/or removed from each manufacturing step. 

	Heparin is a lifesaving drug that was safely used since the 1940s. However, in 2007, contaminated heparin caused a number of deaths in the US and hundreds of adverse reactions worldwide (1). The contaminated heparin was found to contain over-sulfated chondroitin sulfate (OSCS). OSCS was an inexpensive synthetic adulterant that had some anticoagulant activity and was presumably added to heparin to increase profit when the drug was in short supply due to a pig disease outbreak. This “heparin crisis” demonstrated the vulnerability of drug supplies produced from increasingly global manufacturing chains and highlighted the risks inherent in reliance on one country and one animal species as the primary source for a crucial drug.

	To mitigate these concerns by diversifying the sources of heparin drugs, FDA is considering reintroduction of bovine heparin drug product to the US market. In August 2015, the US Pharmacopeial Convention (USP) hosted the 6th Workshop on the Characterization of Heparin Products in São Paulo, Brazil, with co-sponsors, the National Institute for Biological Standards and Control (NIBSC, UK), National Health Surveillance Agency (ANVISA, Brazil), and Sao Paulo State Pharmaceutical Manufacturers Association (SINDUSFARMA, Brazil).  The focus of the workshop was an examination of the global heparin supply chain, specifically the risks of heparin shortages, adulteration, and contamination.

	The following is an overview of scientific research and clinical experience presented at the workshop to generate improved understanding of the differences between porcine and bovine heparins, the clinical implications of reintroducing bovine heparin in the US, and the broader ramifications of bovine heparin in the US market and worldwide.


	Heparin is a natural product, extracted from animals. Just as pork and beef are different from each other, heparin products made from pigs and cattle are similar but not identical. Two sessions of the USP workshop focused on laboratory tests used to understand the differences in structure and biological activity between bovine and porcine heparin. 

	The structure of heparin is that of a linear polysaccharide consisting of repeating disaccharide motifs in which uronic acids alternate with glucosamine. The polysaccharide chains can vary in length and in substitution with sulfates and N-acetyl groups. Structural analysis techniques range from relatively simple, straightforward spectroscopic and chromatographic analyses to sophisticated applications of techniques in nuclear magnetic resonance and mass spectrometry.

	The biological activity of heparin, including its abilities to inhibit the enzymes of blood clot formation 

, can be quantified by several methods. Research suggests that molecular weight and disaccharide composition both play an important role in biological activity. High molecular weight fractions of heparin, for example, have a greater effect than do lower molecular weight fractions on anticoagulation potency.

	Overall, the combination of structural and functional information available provides a clear picture of heparin products from different species. Numerous samples have been tested by heparin manufacturers and academic and regulatory labs revealing clear and consistent differences in the structures (see 

) and biological activity profiles of porcine mucosal and bovine mucosal heparin. In addition, the few samples of bovine lung heparin tested were quite distinct from either mucosal sample type. 

	Importantly, the data presented show that bovine heparin was significantly less potent, weight for weight, than porcine heparin. The relationship between laboratory testing and clinical experience is not straightforward for such complex products, yet a difference in potency could have important clinical relevance (2). Therefore, further investigation including clinical research may be warranted. 

 
	There are two main safety concerns associated with bovine heparin. The first is heparin-induced thrombocytopenia (HIT), an infrequent but potentially devastating adverse event (3, 4). HIT is an immune response in which the body makes antibodies to large complexes formed between the highly sulfated heparin chains and platelet factor 4 (5). HIT occurs in 0.2-5% of patients regardless of the type of heparin administered; porcine and bovine heparin appear similar in terms of HIT risk (6, 7).  Another common adverse event associated with heparin is bleeding, which can be controlled through the neutralization of heparin by protamine sulfate (8). 

	The second safety concern, specific for bovine heparin, is the possible presence of BSE infectious agents (9).  During the BSE epidemic in the UK, some people consumed BSE-infected beef. From 1999-2000, after a long incubation period, some of these individuals developed variant Creutzfeldt-Jakob disease (vCJD); 229 cases have occurred worldwide as of May 28, 2015 (10).  Since its peak in 2000, vCJD has declined significantly but has not been eradicated, as a few cases are still detected every year. No known cases of vCJD, however, have been linked to use of bovine heparin. In addition, in India, Brazil, and Argentina--where bovine heparin products have been in continuous use--no cows have tested positive for BSE and no cases of vCJD have been observed. In the US, only three atypical BSE cases (i.e., different from the distinct BSE strain from the UK that causes vCJD) in cattle have been identified (11).  Since the 1990s, much has been learned about how BSE leads to vCJD in humans. In addition, methods for minimizing BSE risk in bovine materials have advanced (12, 13). Generally, risks from tissue spongiform encephalopathy (TSE) agents are controlled in three steps: animal origin of species and supply chain control, tissue harvest controls, and chemical treatments that remove infectious agents (11).  If bovine heparin is reintroduced, these steps will be instrumental for ensuring patient safety. Because of the safety concerns noted previously, bovine heparin is likely to have its own USP monograph and a separate label (Physician Labelling Rule) that differentiates bovine heparin from porcine heparin.

Possible reintroduction of bovine heparin into the US market


	The only approved source of heparin in most of the world is pig intestine, but the global pig supply is limited geographically. In addition, there is little growth potential for porcine heparin products to be manufactured in other parts of the world. Thus, FDA is concerned about potential shortages due to pig disease or possible geo-political instability. 

	After considering the available options, FDA hosted a meeting with its Science Board in June 2014 to discuss the possible reintroduction of bovine heparin in the US. Reintroduction of bovine heparin would no longer limit the source to one animal species and would extend the geographic distribution of source animals. If disease occurs in one animal source and/or there is geo-political instability in a major source country, the risk of supply shortages could be more readily mitigated.

	The original US-approved heparin drugs from the 1930s were from a bovine source (cow lung) and upon approval of porcine heparin products, both were used interchangeably until the 1990s without major safety risks or concerns. Notably, bovine mucosa heparin drug product is currently available and manufactured in South America (particularly Brazil and Argentina) and India. In some countries, bovine heparin is preferred for religious reasons. Thus, there have been more than 50 years of safe and effective use of bovine lung heparin in patients in the US, and bovine mucosal heparin has been used safely in South America and India. However, because the heparin characterization technology has advanced in the interim, the specifications for the proposed bovine heparin should be modernized in a manner similar to the recent update of the USP porcine heparin monograph.


	Heparin derived from bovine lungs was in general use in Europe and the US until it gradually fell out of use for two reasons: the commercial advantages of the more potent porcine product, and concerns in the 1990s about transmission of BSE to humans through contaminated beef products.

	Workshop speakers from Argentina, Brazil, and India emphasized that bovine heparin has been approved and used for decades in their countries. The Argentinian Pharmacopeia is discussing how to manage bovine and porcine heparins, and the Brazilian Pharmacopeia is currently devising specific monographs for bovine and porcine mucosal heparin.

	The availability of bovine and porcine heparins in Brazil has varied considerably in recent years. In 2008, 42% of heparin was of bovine origin, but this was followed by a decrease and then total removal from the market in 2013. In Argentina, the bovine source accounts for 70% of the total heparin; this has remained unchanged in recent years.

	No serious adverse effects have been associated with the use of bovine heparin in these two countries or in India. Some excess adverse events associated with heparin in cardiovascular surgery, however, were reported to ANVISA in 2008 when, on short notice, bovine heparin replaced porcine heparin for cardiovascular surgery in Brazil. The Brazilian Society of Cardiovascular Surgery (14) and ANVISA (15) published warning notes suggesting careful monitoring of anticoagulant levels when using bovine heparin.

	There are no reports of clinical trials comparing heparins from bovine versus porcine intestine. Therefore, although bovine heparin has been used successfully in several countries for many years, caution may be required when porcine heparin is replaced with bovine heparin without warning.

Bovine heparin use in the manufacture of LMWHs


	Low molecular weight heparins (LMWHs) are manufactured from heparin sodium (also known as unfractionated heparin sodium, or UFH) using chemical or enzymatic depolymerization methods (16). Currently, in the US, all forms of LMWH are made from porcine UFH. Therefore, their composition and properties are known based on their biological starting material. 

	The structure and composition of bovine UFHs differ from that of porcine; therefore, a LMWH product made from bovine UFH could have different properties from the same product made from porcine UFH. For example, if enoxaparin (a LMWH) is produced from bovine heparin in the future, this product could not be called enoxaparin because the structure and properties would be different and the activity would probably be different as well. 


	The current USP Heparin Sodium monograph describes system suitability and acceptance criteria for identity, purity, and strength that are designed strictly for porcine heparin (17, see 

	The workshop presentations showed clear differences in the structures and biological activity profiles of porcine mucosal, bovine lung, and bovine mucosal heparin samples, thereby suggesting the need to have a separate monograph for bovine heparin sodium. The structural differences would necessitate separate identification reference standards (RS) for 1H NMR, chromatographic identity, and molecular weight determination tests for bovine heparin (see Table I). Depending on the tissue source(s) of bovine heparin, there may be a need to establish separate identification RS for bovine lung and bovine mucosal heparins.  

	Based on the testing of numerous samples, the anticoagulant activity of bovine heparin is significantly lower than that of porcine in the laboratory. Most of the current bovine products gave potencies of about 100 IU/mg and some batches were estimated to be as low as 70 IU/mg.  Considering the monograph specification for porcine heparin is 180 IU/mg, it is likely that the clinicians will need to give more bovine heparin than porcine heparin by weight. Many years of safe bovine heparin use suggest that the higher amounts needed, as compared with porcine heparin, do not impact clinical efficacy. Clinical issues with bovine heparin, however, will need to be monitored carefully because wider clinical use could identify unforeseen differences between porcine and bovine heparin. Furthermore, bovine heparin requires higher doses of protamine for neutralization.

	Future bovine heparin production sites (both drug substance and drug product) should be under cGMP compliance. Supply chains including farms, slaughterhouses, and facilities that isolate, treat, store, and ship the bovine tissue need to follow the same steps and tests described for porcine heparin (18). These steps include, but are not limited to:

		Determine the species origin to verify that the ingredient comes only from the correct species.

		Confirm the absence of OSCS and ruminant material contaminants from another species. 

		Ensure that all heparin suppliers are audited and inspected regularly regarding their documentation practices and compliance with cGMP. 

		Reject any lots containing mucosa from another species.


	Heparin is an essential, life-saving drug that is needed worldwide. To avoid drug shortages, FDA is considering reintroduction of bovine heparin, which would diversify the supply chain by adding a bovine source to the currently used porcine heparin. Sourcing heparin from two species could greatly reduce vulnerability to shortages when disease strikes one species, and could also reduce reliance on one country as the primary source. The risks involved in reliance on one species from one country were clearly illustrated by the heparin crisis of 2007-2008, when adulterated porcine heparin from China caused numerous deaths and hundreds more adverse effects (1). Currently, China is the source for roughly 60% of crude porcine heparin used in the US and Europe.

	Bovine heparin is currently being used in some countries and was used safely for more than 50 years in the US before manufacturers voluntarily withdrew it from the market during the BSE crisis in the UK. Despite concerns about bovine products, there are no known cases of BSE contamination of bovine heparin. If bovine heparin is reintroduced in the US, methods for inactivating BSE could be applied to further reduce any risk. The other main safety issue with heparin is a severe adverse effect called HIT, but bovine heparin does not appear to have higher rates of HIT than does porcine heparin.

	Porcine and bovine heparins are distinctly different in terms of their structures and biological activities. These complex products may behave differently in clinical use than in laboratory testing, but if potency does in fact differ significantly in clinical use, this will need careful evaluation and perhaps dosage adjustment to avoid giving patients too much or too little heparin. If bovine-sourced heparin is reintroduced, supply-chain control will be critical, with frequent inspections of slaughterhouses and processing facilities for cGMP compliance. From the data presented at the meeting, bovine heparin and porcine heparin are not equivalent drugs, and therefore, they will require two different compendial monographs. The next steps are for manufacturers to bring bovine products to the regulatory agencies for evaluation and possible reintroduction to the market after a 15-year absence. 


	The participants in the USP 6th Workshop on the Characterization of Heparin Products are acknowledged with gratitude. 

, 19, 921-933 (2010).
	2. A. Tovar et al., 

 6, 230 (2013)
	3. T.E. Warkentin et al., 

106, 3791-3796 (2005).
	4. T.E. Warkentin and A. Greinacher, 

76, 2121-2131 (2003).
	5. A. Greinacher et al., Arterioscler Thromb Vasc Biol 26, 2386-2393 (2006).
	6. J. E. Ansell et al., 

 88, 878-882 (1985).
	7. J.L. Francis et al., 

 75, 17-22 (2003).
	8. G. Costantino et al., 

 7, e44553 (2012).
	9. J.L. Harman and C.J. Silva, 

Journal of the American Veterinary Medical Association

 234, 59-72 (2009).
	10.   EuroCJD Network, 

Creutzfeldt-Jakob Disease International Surveillance Network

 (2015).
	12. I. DeVeau et al., “Analytical Microbiology Expert Committee. The USP Perspective to Minimize the Potential Risk of TSE Infectivity in Bovine-Derived Articles Used in the Manufacture of Medical Products.,” 

 30, 1911-1921 (2004).
	13. D. Taylor,  

 325, 75-76 (2002).
	14. W. J. Gomes and D.M. Braile. 

 (2008), , accessed Aug. 27, 2015.
	16. H. Liu et al., 

, First Supplement, Heparin Sodium monograph, p.3748 (USP, Rockville, MD).
	18. FDA, 

Heparin for Drug and Medical Device Use:  Monitoring Crude Heparin for Quality


	This article reflects the views of the authors and should not be construed to represent US FDA’s views or policies.


	David Keire is acting laboratory chief, Branch I, Division of Pharmaceutical Analysis, FDA.
	Barbara Mulloy is visiting professor at Institute of Pharmaceutical Sciences, King’s College London.
	Christina Chase is senior scientific writer at US Pharmacopeial Convention (USP).
	Ali Al-Hakim is acting director of Division of New Drug API at the FDA.
	Damian Cairatti is head of Latin American Regulatory Affairs at USP.
	Elaine Gray is principal scientist at National Institute of Biological Standards and Control (NIBSC) in the UK.
	John Hogwood is research scientist at NIBSC.
	Tina Morris is senior VP of Science Global Biologics at USP.
	Paulo Mourão is professor at Federal University of Rio de Janeiro.
	Monica Da Luz Carvalho Soares is a member of the Deliberative Council of the Brazilian Pharmacopeia at ANVISA and a visiting fellow at the University of Maryland Baltimore County (UMBC).
	Anita Szajek is principal scientific liaison at USP.

 When referring to this article, please cite it as D. Keire et al., “Diversifying the Global Heparin Supply Chain: Reintroduction of Bovine Heparin in the United States?,” 

Articles

The global supply chain for bovine and porcine heparin and regulatory considerations are examined.