462274

2012

PRF28110.1177/0267659112462274PerfusionHedlund KD et al.

The heparin recall of 2008

KD Hedlund,1 DP Coyne,2 DM Sanford3 and J Huddelson4

Perfusion 28(1) 6165 The Author(s) 2012 Reprints and permission: sagepub. co.uk/journalsPermissions.nav DOI: 10.1177/0267659112462274 prf.sagepub.com

Abstract Heparin is one of the oldest drugs still in widespread clinical use. Its discovery in 1916 predates the establishment of the Food and Drug Administration. Since 1935, over 15,000 research papers have been published on the effects of heparin. Because the exact chemical formula of heparin is unknown, synthetic manufacturing has proven difficult. In 2008, a worldwide recall of heparin occurred. Scientists determined that a contaminant known as oversulfated chondroitin sulfate was responsible for the numerous deaths and adverse events. This contaminant was first traced to a chemical plant in Changzou, China. This article will review the discovery of heparin and the adulteration process that jeopardized the worlds heparin supply. Keywords Heparin; heparin contamination; anticoagulant; cardiopulmonary bypass; antithrombin III

Introduction
Heparin, a highly sulfated glycosaminoglycan, is not a singular substance. Rather, it is a complex heterogeneous mixture of straight-chain polysaccharide molecules, varying in both sequence and number. Owing to its negative charge, heparin is often referred to as an anionic polyelectrolyte.1 Alone, heparin has very little effect on coagulation. In the blood, however, the combination of heparin and antithrombin, a serine protease inhibitor, greatly increases the rate of inhibition of two principle procoagulant proteases Factor Xa and Factor IIa (thrombin). This conformational shape change in the C-terminal portion of the antithrombin molecule is induced through a specific pentasaccharide sequence found only in a third of heparin chains (see Figure 1). While Factor Xa is inhibited by virtually all active fractions of heparin, thrombin inhibition by the heparin/ antithrombin complex requires a specific thrombinbinding domain found only on heparin fragments of 18 polysaccharide units or longer.2 The molecular weight of unfractionated heparin is 5,000 to 40,000 Daltons, although, in the vast majority of preparations, the predominant range is 12,000 to 15,000 Daltons. At the present time, heparin is the best anticoagulant for procedures involving extracorporeal circulation. It is efficient at preventing thrombus formation, even in static areas such as the cardioplegic delivery line. Its effect can be measured by using the simple accelerated clotting time test, and protamine sulfate acts as a fast and reliable antidote when reversal is desired. Heparin is one of the oldest drugs still in widespread clinical use. Its discovery predates the establishment of the Food and Drug Administration (FDA). More than 15,000 research papers dealing with heparin have been published since 1935. Over a trillion units of heparin are administered to patients in the United States each year equating to approximately 24 tons. Along with antitoxins, heparin is one of the few drugs that cannot be effectively synthesized and is currently prescribed for the treatment of human disease. The process of preparing pharmaceutical grade heparin has been altered over time, as the primary tissue source has changed from dog liver to beef lung and, finally, to porcine intestine. Virtually all of worlds heparin is derived from porcine mucosa, scraped from the degranulating mast cells that line the intestines. Some preparations, however, utilize the whole intestine (hashed pork guts). These two raw materials contain differing amounts of heparan sulfate, the structurally related molecule that exists on the surfaces of vascular endothelial cells and leukocytes.3

1The

Michael E. DeBakey Heart Institute, Hays Medical Center, Hays, Kansas, USA 2Kansas Medical Center, Andover, Kansas, USA 3Jefferson Regional Medical Center, Pine Bluff, Arkansas, USA 4Self Regional Hospital, Greenwood, South Carolina, USA Corresponding author: Kelly D. Hedlund The Michael E. DeBakey Heart Institute Hays Medical Center 2220 Canterbury Drive Hays, Kansas 67601, USA Email: khedlund@haysmed.com

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Figure 1. Only a third of heparin chains exhibit the unique pentasaccharide configuration necessary for binding antithrombin.

Also, the heparins lot-to-lot variability is due to the environment and diet that each species of pig is exposed to prior to slaughter. Heparin has been obtained from other animal sources, such as whales, mice, lobsters and camels.

Discovery and purification of heparin

Heparin was first discovered in 1916 by Jay McLean, a medical student at Johns Hopkins Medical School. McLean, on the order of his supervising professor, William Howell, was actually in search of a coagulant, not an anticoagulant. This was the era when hemorrhage, a common complication of accidents, war wounds and even childbirth, was considered the great killer of man. McLeans incidental discovery, an extract from a dogs liver, was called hepar phosphatid. When mixed with a beaker of cats blood, the extract prevented clotting for hours. In a paper published by Howell in 1918, McLeans extract was renamed heparin and its anticoagulant properties made public for the first time.4 Unfortunately, when McLeans heparin was injected into animals, the result was fever, shock and death. Dismayed and dejected, McLean left Johns Hopkins in 1924. Continuing the research, Howell proposed using heparin as an anticoagulant for bottled human blood in 1928. Again, fever and shortness of breath accompanied the administration of this blood to patients. Alas, McLeans discovery, considered unsafe for human use, was relegated to the dusty shelves of Howells laboratory, seemingly, forever. In 1929, however, a Canadian researcher named Charles Best took up the charge of studying heparin. Best was already famous for co-discovering insulin in 1921 (with Frederick Banting). In the early 1930s, Best determined that heparin could be extracted from other animal organs besides the liver. Through painstaking work, his team in Toronto successfully extracted heparin from beef lung, beef intestine and pork intestine. By 1935, a purified version of heparin, devoid of toxic effects, was made available for human use in Canada and

Europe.5 Gordon Murray, a thoracic and vascular surgeon at Toronto General Hospital, is the first-known physician to successfully prescribe heparin for the prevention of deep vein thrombosis (DVT) following surgery. Commercially available heparins began to appear in 1937. In the United States, Dr. Conrad Lam and Dr. Ray McClure, both of the Ford Hospital in Detroit, are credited with first using heparin, in 1939, to treat DVT and pulmonary embolism.

Heparin contamination
In November 2007, healthcare workers in St. Louis, Missouri observed an allergic-type reaction in a six year-old child undergoing dialysis.6 Two days later, another child in the same pediatric dialysis unit exhibited similar symptoms stridor, nausea, hypotension, tachycardia and swelling of the lips and face. Both the FDA and the United States Centers for Disease Control and Prevention were notified. Within days of this notification, responses began pouring in from other dialysis units around the United States of similar patient reactions. In response, Baxter Healthcare Corporation recalled nine lots of its heparin injection and multi-dose vials on January 17, 2008.7 At the time, Baxter was the distributor of nearly half the supply of heparin in the United States and globally. On February 28, 2008, with drug production temporarily halted, Baxter recalled all remaining lots of its heparin supply. Following the recall, FDA officials traveled to China to inspect facilities where the Baxter heparin was manufactured. Concomitantly, an international consortium of experts was enlisted to work with the FDA and the pharmaceutical industry to elucidate the problem. On March 19, 2008, investigators reported that a heparin-like contaminant known as oversulfated chondroitin sulfate (OSCS) was the likely cause of the adverse reactions.8 Furthermore, the FDA contended that OSCS was made in China from animal cartilage, that it was chemically altered to act like heparin and that it was added intentionally to make production of heparin cheaper.

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Initially, OSCS was traced to a specific chemical plant in Changzhou, China a facility owned, in part, by Scientific Protein Laboratories, LLC of Waunakee, Wisconsin. Interestingly, Scientific Protein Laboratories was founded in 1976 by the famed hot dog maker, Oscar Meyer. Since then, however, the FDA has identified at least 12 other Chinese companies that produced tainted heparin, with dates of manufacture going back to 2006.9 About this same time, Asian and European authorities observed similar allergic reactions in patients receiving heparin manufactured in Japan and Germany, respectively. At present, samples containing OSCS have shown up in at least 12 countries around the world.10 According to the FDA, over 200 deaths and thousands of adverse events have been reported in the United States alone since Baxters initial recall. Voluntary recalls were subsequently issued by three other heparin suppliers (American Health Packaging, B. Braun Medical and Covidien), despite not receiving reports of adverse events. On April 8, 2008, simultaneous on-line publications confirmed that OSCS was the adulterant responsible for the heparin-related adverse events.11,12 The FDA further issued warnings to manufacturers of heparin-coated products, such as heart stents, vascular grafts and perfusion devices. On May 8, 2008, the FDA sent a letter to Celsus Laboratories of Ohio, a distributor of both heparin sodium and heparin lithium, urging them to inform their customers of the recall. Knowing that Celsus imported heparin ingredients from China, the FDA had performed an on-site inspection a month prior. Citing insufficient compliance, the FDA conducted a repeat inspection of Celsus Cincinnati facility and, in November 2008, seized 11 lots of heparin contaminated with OSCS.

properties similar to heparin (see Figure 2). This explains why traditional batch testing for heparin potency did not identify OSCS as a contaminant. Because CS is an animal derivative, it is cheap and abundant. Synthesizing CS into OSCS is a relatively easy process. Unfortunately, OSCS is neither benign nor innocuous in its action. For instance, it activates the kinin-kallikrein pathway, leading to the generation of bradykinin, a potent vasoactive mediator.14 In addition, it activates the complement system, resulting in anaphylaxis-like reactions, such as facial swelling, tachycardia, nausea and hypotension. These harsh side effects mimic the anaphylaxis-like reactions seen in the thousands of affected patients who received China-supplied heparin. The origins of OSCS appear to date back to 1969 when the Seikagaku Kogyo Company of Japan received a United States patent for the production of chrondroitin polysulfate. In 1985, the Luitpold-Werk Pharmaceutical Company of Germany successfully produced a polysulfated chondroitin drug called Arteparon. The drug was marketed in Europe as an anti-arthritic, but was subsequently pulled from the market in 1992 due to reports of allergic-type reactions and cross-reactivity with heparin. In 1995, a Frenchman named Petiou received a United States patent for sulfated glycosaminoglycanoid derivatives of the dermatan sulfate and chondroitin sulfate

Sulfonation of chondroitin sulfate

Chondroitin sulfate (CS) is a naturally-occurring dietary supplement. Like heparin, the backbone of CS is comprised of repeating disaccharide units. It is a major component of cartilage and its chemical formulation generally includes 11.5 sulfate groups per saccharide molecule. Comparatively, most heparin chains have an intermediate level of sulfonation; that being 22.5 sulfate groups per saccharide unit. Given that heparin comes from animals, the potential for impurities is large, even in the finished product. The most prevalent impurity is dermatan sulfate (DS). Similar to heparin, DS contains iduronic acid as part of its disaccharide chain, although its negative charge density is much lower. Levels of DS commonly found in pharmaceutical grade heparin range from 1-7%.13 The contaminant OSCS, sometimes referred to as hyper-sulfated chondroitin sulfate, is not naturally occurring. It must be chemically modified from CS in order to exist. With four attached sulfate groups, the OSCS molecule is highly negatively charged, thus, exhibiting anticoagulant

Figure 2. Comparison of glycosaminoglycans that exhibit disaccharide units similar to heparin (a: chondroitin sulfate; b: dermatan sulfate; c: oversulfated chondroitin sulfate).

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type. Then, in 1998, researchers from the University of Iowa and Japan collaborated to produce a synthetic heparin, using the chondroitin sulfate sulfonation process.15 Following their initial publication, the researchers administered their highly sulfated anticoagulant to mice. The mice all died. Unfortunately, no follow-up report was published, as the experiment was deemed unsuccessful and unworthy of peer review. On December 20, 2005, Shandong University of China applied for a patent for the preparation of OSCS. Within weeks of the patent being filed, chemists created batches of OSCS which, presumably, found their way into the Chinese heparin supply. Only scant amounts of OSCS were found in heparin samples manufactured in early 2006. However, by late 2007, the levels of OSCS had risen to as high as 35%.16

Industry reactions
The United States Pharmacopeia (USP) first published a monograph on heparin in 1950. Historically, heparins potency has been standardized using a clotting assay comprised of sheeps plasma. A crude flame test for the presence of sodium was also used for drug identification. In 1983, a potency test for anti-Factor Xa was added. In response to the heparin recall in early 2008, new quality control measures, such as nuclear magnetic resonance spectroscopy and anion exchange chromatography, were adopted to help identify impurities and ensure against adulteration. In addition, the USP replaced the sheep plasma assay with a chromogenic anti-Factor IIa assay. A second step taken by the USP was to calibrate their potency reference standard with the international standard issued by the World Health Organization. In short, this alignment, announced on August 21, 2009, resulted in an approximate 10% decrease in the potency of USP heparin.17 These necessary revisions took effect on October 1, 2009.

domain, using sophisticated chromatography techniques.19 In 1989, chemists produced more than 20 grams of a synthetic pentasaccharide drug for toxicology studies in healthy volunteers. In 2001, a commercial version of this heparin derivative was released to European and United States markets under the name Arixtra (Fondaparinux). Unfortunately, over 50 steps are required in the chemical synthesis, and the cost is 2 to 3 times that of LMWHs and, as with LMWHs, the pentasaccharide analogues exhibit anti-Xa activity, but show little or no inhibition of thrombin. Moreover, unlike unfractionated heparin, the anticoagulant activity of these pentasaccharides cannot be reversed with protamine. Drawbacks such as these strongly suggest that synthetic heparins of the pure pentasaccharide type will never replace unfractionated heparin. Chemoenzymatic processes for manufacturing synthetic heparin appear promising. Researchers, using fermented bacterial products such as Escherichia Coli, bioengineered heparin in as few as six steps.20 Remarkably, the antithrombin binding, anti-Factor Xa, and anti-Factor IIa activities appear comparable to todays unfractionated heparin. It remains to be seen, however, if large-scale quantities can be produced affordably to meet worldwide demand. Until then, its worth remembering that heparin, a drug used for over 50 years, is still largely misunderstood.
Funding
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Conflicts of Interest Statement

The authors declare that there are no conflicts of interest.

References
1. Bigelow WG. Mysterious heparin: the key to open heart surgery. Toronto: McGraw-Hill Ryerson, 1990. 2. Despotis GJ, Joist JH. Anticoagulation and anticoagulation reversal with cardiac surgery involving cardiopulmonary bypass: an update. J Cardiothorac Vasc Anesth (Suppl 1) 1999; 13: 1829. 3. Bishop JR, Schuksz M, Esko JD. Heparan sulphate proteoglycans fine-tune mammalian physiology. Nature 2007; 446: 10301037. 4. Howell WH, Holt B. Two new factors in blood coagulation heparin and pro-antithrombin. Amer J Physiol 1918; 47: 328341. 5. Best CH. Preparation of heparin and its use in the first clinical case. Circulation 1959; 19: 7986. 6. Quinn K. St. Louis hospitals help uncover batches of contaminated heparin. www.ksdk.com 04/30/2008. 7. Baxter Healthcare Corporation Urgent Product Recall January 17, 2008. 8. Metzger LC. The scoop on heparin. www.oley.org 04/10/2008.

Future developments
The obvious alternative that comes to mind when looking for new ways to produce safe heparin is chemical synthesis. This route is difficult and expensive, however, especially for larger heparin saccharides. In the early 1980s, researchers successfully produced low molecular weight heparins (LMWH) through a controlled enzymatic fragmentation process.18 While efficacious in the prevention of deep vein thrombosis, LMWH chains are generally too short to inhibit thrombin via the ternary bridging complex. Thus, LMWHs affect the coagulation cascade primarily through inhibition of Factor Xa, making them impractical for use in extracorporeal circulation procedures. About this same time, researchers elucidated the unique structure of the heparin pentasaccharide

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9. Powell B. Heparins deadly side effects. www.time.com 11/13/2008. 10. Harris G. Problems in blood drug lead to halt by factory. www.nytimes.com 02/12/2008. 11. Kishimoto TK, Viswanathan K, Ganguly T, et al. Contaminated heparin associated with adverse clinical events and activation of the contact system. N Engl J Med 2008; 358: 24572467. 12. Guerrini M, Beccati D, Shriver Z, et al. Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events. Nature Biotechnol 2008; 26(6): 669675. 13. Beni S, Limtiaco JFK, Larive CK. Analysis and characterization of heparin impurities. Anal Bioanal Chem 2011; 399: 527539. 14. Liu H, Zhang Z, Linhardt RJ. Lessons learned from the contamination of heparin. Nat Prod Rep 2009; 26: 313321.

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15. Maruyama T, Toida T, Imanari T, Yu G, Linhardt RJ. Conformational changes and anticoagulant activity of chondroitin sulfate following O-sulfonation. Carbohydr Res 1998; 306: 3543. 16. Royce K. FDA failures contribute to spread of contaminated drugs. www.worldfocus.org 12/17/2008. 17. United States Pharmacopeia Statement. Heparin potency unit assignment and harmonization with the international standard for unfractionated heparin. Release date August 21, 2009. 18. Barrowcliffe TW. History of heparin. Handb Exp Pharmacol 2012; 207: 322. 19. Petitou M, van Boeckel CA. A synthetic antithrombin III binding pentasaccharide is now a drug: what comes next? Angew Chem Int Ed 2004; 43: 31183133. 20. Turnbull JE. Getting the farm out of pharma for heparin production. Science 2011; 334: 462463.

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