API Update: Heparin Scare a Lesson in Safeguarding APIs

By Catherine Shaffer In 2007 and 2008, patients—primarily those undergoing hemodialysis—began having severe, life-threatening allergic reactions to a drug that is normally very safe—heparin. The reactions were at first quite mysterious.1 As adverse-event reports piled up and some patients died, drug manufacturers, suppliers, and regulatory agencies joined forces to track down and identify the contaminant.

In addition to increased testing of heparin and inspection of factories, the FDA has taken a number of steps to prevent contamination, not only with heparins but also with other active pharmaceutical ingredients vulnerable to contamination.

Early in 2008, heparin manufacturer Baxter International Inc. issued a voluntarily recall of nine lots of heparin.2 Because heparin is a life-saving drug for some patients and the contamination was so widespread, it was not possible to immediately remove all affected product, and doctors were put in the agonizing position of dispensing a drug with known contamination.
Low molecular weight heparin products were also affected, leading to recalls of Lovenox (enoxaparin sodium) by Sanofi-Aventis and heparins made by France’s Rotexmedica and Italy’s Opocrin S.p.A.
Ultimately, the primary contaminant was identified as oversulfated chondroitin sulfate (OCS).3-5 Because that chemical does not occur in nature, and because it shares some properties with heparin, it is thought to be an example of economically motivated adulteration (EMA).6 The discovery of melamine in milk products and pet foods is another example. Perpetrators of EMA add a foreign substance in order to counterfeit a product or enhance a poor one. EMA is a huge problem for products sourced from China, as is increasingly the case in the drug industry.
Three years after the recall, the drug industry and the FDA have developed regulations and protocols for testing heparin sources. While steps have been taken to make it more unlikely that contaminated heparin will slip past U.S. agencies again, the greater question is how many other products could be affected by economically motivated adulteration. In theory, any complex biological mixture could contain difficult-to-detect contaminants.

Rapid Tests for OCS

German researchers combined a two-step fluorescence assay and a two-step anti-Factor Xa assay to detect counterfeit heparin and protein in heparin. And researchers from Sanofi-Aventis in Paris developed a polymerase chain reaction method for quality control of heparins.

One of the scientific community’s first responses was to develop workable methods for testing heparin sources to confirm the purity of the product. During the crisis, a number of labs worked together to use sophisticated methods like nuclear magnetic resonance (NMR), heteronuclear single quantum coherence (HSQC), total correlation spectroscopy (TOCSY), and high performance liquid chromatography (HPLC) to analyze the complex biological samples. Unfortunately, those methods are not easily available to the agencies charged with inspecting factories or testing product.
German researchers combined a two-step fluorescence assay and a two-step anti-Factor Xa assay to detect counterfeit heparin and protein in heparin.7 And researchers from Sanofi-Aventis in Paris developed a polymerase chain reaction method for quality control of heparins.8
Researchers at Rensselaer Polytechnic Institute are developing hyphenated methods of analyzing heparins. They studied combinations of numerous analytical techniques, including mass spectrometry, HPLC, capillary electrophoresis, and NMR spectroscopy to find the best methods for analyzing heparin, concluding that a combination of separation and spectra techniques is necessary for the best results.9

The FDA Acts

In addition to increased testing of heparin and inspection of factories, the FDA has taken a number of steps to prevent contamination incidents, not only with heparins but also with other active pharmaceutical ingredients (APIs) vulnerable to contamination.
The FDA’s inspection process now takes into account traceability, testing, verification of controls, and supplier qualification. The agency is also developing risk models to predict which drugs and ingredients are most at risk of adulteration in order to develop a response before a crisis emerges.
In a letter to the FDA, Martin VanTrieste, vice president of quality and commercial operations at Amgen, encouraged the FDA to “think like a criminal would” to counter attempts at deliberate adulteration motivated by opportunities such as supply shortages (M. VanTrieste [mvantrie @amgen.com], e-mail, May 1, 2009).
“We know that the shortage of pigs in China provided an opportunity for unethical players and criminals to introduce economically motivated adulterated heparin into the supply chain. Learning from this lesson, does it apply to today, where we face a potential pandemic flu with a shortage of effective anti-viral agents that governments around the world are trying to stockpile?” wrote VanTrieste.
The FDA has ranked more than 1,000 APIs in order of risk of EMA, based on their multifactorial model, and have targeted a subset of high-risk ingredients for additional sampling and testing at the border.
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Case study: The Hunt for a Killer Contaminant

A large team of experts assembled to carry out the analysis of contaminated lots of heparin, including scientists from the Massachusetts Institute of Technology in Boston, Momenta Pharmaceuticals Inc. in Cambridge, Mass., Rensselaer Polytechnic Institute in Troy, N.Y., and the G. Ronzoni Institute for Chemical and Biochemical Research in Milan.
The lots involved in the reactions had already been tested for typical contaminants such as proteins, lipids, and DNA, and nothing suspicious had been found in them. Tests for atypical contaminants such as lead and dioxin also revealed nothing. “My first instinct in looking at it was that it was an accidental, not a deliberate, contamination,” said Robert Linhardt, PhD, a professor of chemistry at Rensselaer Polytechnic Institute and a noted heparin expert. “We didn’t assume instantly that there was some nefarious activity here ... . Most problems with pharmaceuticals are just that—accidental.”
Because heparin is a complex mixture of biomolecules, the strategy for analyzing it involved a combination of orthogonal techniques. Nuclear magnetic resonance (NMR) analysis revealed a string of unusual N-acetyl signals, which suggested O-substituted N-acetylgalactosamine. Analysis by heteronuclear single quantum coherence (HSQC), total correlation spectroscopy (TOCSY), correlation spectroscopy (COSY), and rotating-frame nuclear Overhauser enhancement spectroscopy (ROSEY) supported that finding. It was now known that the contaminant was a polymeric repeat of N-acetylgalactosamine linked to glucuronic acid exclusively through beta linkages.
High performance liquid chromatography (HPLC) analysis of digests with heparinases or heparinases plus delta-4,5 glycuronidase and 2-O sulfatase were consistent with the NMR and other results. Further analysis of the isolated contaminant by NMR and other methods yielded a match for oversulfated chondroitin sulfate.
Once the unknown contaminant was identified as oversulfated chondroitin sulfate, it was positively connected with the adverse clinical symptoms that had been observed. Because oversulfated chondroitin sulfate is not found in nature, researchers concluded that its presence in the lots of heparin was no accident, as Dr. Linhardt at first assumed. It was a case of deliberate product adulteration.

Companies’ Efforts

“Mostly, after the recall, people didn’t want to deal with China at all,” Brian James, PhD, director of strategic development at the pharmaceutical consulting firm Rondaxe, told PFQ. “They were going to stay away from China at all costs.” Rondaxe, in Syracuse, N.Y., advises companies in the pharmaceutical and biotechnology industry on issues related to manufacturing, business development, and information technology. In the aftermath of the heparin recalls, Dr. James worked with a number of companies to secure API supplies sourced from overseas.

“Product is the process. When you globalize those types of drugs, it’s difficult to be certain that the process is well controlled. You can’t just do it by depending on analysis.”

—Robert Linhardt, professor of chemistry, Rensselaer Polytechnic Institute, Troy, N.Y.

Dr. James cited language as one of the biggest issues in working with a supplier in China. He advised bringing your own translators rather than relying on those provided by the company you are inspecting. A fluent speaker of Mandarin can pick up on sketchy conversations or inaccurate documentation.
Another factor that threatens purity of APIs is the intertwining of food supply and drug supply chains. Heparin is sourced from pig intestines. Pig farming in China is in no way regulated or inspected by the FDA, so even the most scrupulous supplier can’t guarantee that the pigs have been handled in a manner consistent with American current good manufacturing practice standards.
“Product is the process,” said Robert Linhardt, PhD, a professor of chemistry at Rensselaer Polytechnic Institute in Troy, N.Y., and a noted heparin expert. “When you globalize those types of drugs, it’s difficult to be certain that the process is well controlled. You can’t just do it by depending on analysis.”
Dr. Linhardt’s lab is working to develop a non-animal source of heparin.
Experts agree that responsibility for purity of product and source ingredients falls on the manufacturer, not the FDA or any other intermediary. In an increasingly globalized economy where the rules of engagement vary drastically among cultures, it is important to think ahead and not get caught flat-footed by an instance of EMA. The best defense is to “think like a criminal” and anticipate how a product could be adulterated or counterfeited before it happens.

References

1. Centers for Disease Control and Prevention. Acute allergic-type reactions among patients undergoing hemodialysis—multiple states, 2007-2008. MMWR Morb Mortal Wkly Rep. 2008;57(5)5:124-125. Available at: www.cdc.gov/mmwr/preview/mmwrhtml/mm5705a4.htm. Accessed February 13, 2011.
2. U.S. Food and Drug Administration (FDA). Baxter issues urgent nationwide voluntary recall of heparin 1,000 Units/ml 10 and 30ml multi-dose vials. FDA website. January 25, 2008. Available at: www.fda.gov/Safety/Recalls/ArchiveRecalls/2008/ucm112352.htm. Accessed February 13, 2011.
3. Guerrini, M, Beccati D, Shriver Z, et al. Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events. Nat Biotechnol. 2008;26(6):669-675. Available at: www.nature.com/nbt/journal/v26/n6/abs/nbt1407.html. Accessed February 13, 2011.
4. 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(23):2457-2467. Available at: www.nejm.org/doi/pdf/10.1056/NEJMoa0803200. Accessed February 13, 2011.
5. McMahon AW, Pratt RG, Hammad TA, et al. Description of hypersensitivity adverse events following administration of heparin that was potentially contaminated with oversulfated chondroitin sulfate in early 2008. Pharmacoepidemiol Drug Saf. 2010;19(9): 921-933. Available at: http://onlinelibrary. wiley.com/doi/10.1002/pds.1991/abstract. Accessed February 13, 2011.
6. U.S. Food and Drug Administration (FDA). Margaret A. Hamburg, M.D., Commissioner of Food and Drugs - remarks at the partnership for safe medicines interchange 2010. FDA website. October 8, 2010. Available at: www.fda.gov/NewsEvents/Speeches/ucm229191.htm. Accessed February 13, 2011.
7. Alban S, Lühn S, Schiemann S. Combination of a two-step fluorescence assay and a two-step anti-Factor Xa assay for detection of heparin falsifications and protein in heparins. Anal Bioanal Chem. 2011;399(2): 681-690. Available at: www.ncbi.nlm.nih.gov/pubmed/20953779. Accessed February 13, 2011.
8. Auguste C, Dereux S, Martinez C, et al. New developments in quantitative polymerase chain reaction applied to control the quality of heparins. Anal Bioanal Chem. 2011; 399(2):747-755. Available at: www.springerlink.com/content/303196016213l163/. Accessed February 13, 2011.
9. Yang B, Solakyildirim K, Chang Y, et al. Hyphenated techniques for the analysis of heparin and heparin sulfate. Anal Bioanal Chem. 2011;399(2):541-557. Available at: www.springerlink.com/content/973281mj11796736/. Accessed F