Early this year, as we all stood riveted and perplexed by the news that patients had died after receiving contaminated heparin, FDA and key members of the scientific community scrambled to find answers and uncover the roots of the tainted product. Led by Moheb Nasr, PhD, director of FDA’s Office of New Drug Quality Assessment (ONDQA), the group traced the contaminant to one of Baxter’s suppliers in China, and to a contaminant known as over-sulfated chondroitin sulfate. The following is a behind-the-scenes look at these forensic endeavors.
Note: This article is part of a larger feature, “Lessons from Heparin,” which appeared in the September issue of Pharmaceutical Manufacturing magazine.
The detective work required to determine the contaminant was challenging, particularly since there was no developmental data available on the drug, says Moheb Nasr, PhD, director of FDA’s Office of New Drug Quality Assessment (ONDQA), whose team had to, essentially, recreate the “Quality by Design” data for heparin. “We had to start from scratch, conducting very detailed quality risk assessment of all aspects of the manufacturing process and the product characterization in order to identify what could be the potential cause or causes of this problem,” he says. A systematic, scientific approach was prudent rather than “shooting in the dark” with different analytical methods, Nasr adds.
The analytical sleuthing to determine the nature and source of the heparin contaminant was impressive. Ram Sasisekharan, PhD, from MIT, at the behest of the FDA (in the person of Moheb Nasr), spearheaded a joint effort to discover what, if anything, was wrong with a number of lots of heparin distributed by Baxter Healthcare.
Work had already been started by Dr. Nasr’s group at CDER, who had already used capillary electrophoresis (CE) and heparinases; they demonstrated that something was in the samples that did not qualify as heparin. He asked Sasisekharan to qualify exactly what the adulterant(s) was. One problem, of course, is that heparin is derived from pigs, and natural products are never exactly the same.
Heparin is mostly a polymer of disaccharides (two-ring sugars) with each unit containing uronic acid and glucosamide. When the various stereoisomers, sugars and sulfation patterns are combined, there are potentially 32 disaccharide units to be included in what is labeled “heparin.” The size of the task suggested two more research groups be included: the Giuliana Ronzoni Institute for Chemical and Biochemical Research (Milan), which had been working with heparin for some time, and Momenta Pharmaceuticals (Cambridge, Mass.), equipped with state-of-the-art high throughput analysis equipment to perform numerous analyses in parallel.
The work began with 10 samples from FDA: six determined to be adulterated and four “clean” controls. These were blinded by the MIT researchers and sent for analysis. Preliminary work with 1H and 13C (one dimensional) NMR yielded some promising results:
- 1H NMR showed a peak at 2.16 ppm, signaling an acetyl group, determined to be not from heparin (2.06 ppm) or the common contaminant dermatan sulfate (2.08 ppm).
- 13C NMR gave unusual peaks at 25.6 ppm and 53.5 ppm. These uncovered the presence of an O-substituted N-Acetylgalactosamine.
- 13 C also showed a signal between 103 and 105 ppm that implied a β-glycoside linkage between monosaccharides.
When two-dimensional NMR (plotting spectrum/spectra from one technique versus another) was tried, even more information was gleaned. Both sugars in the disaccharide unit contained two sulfate groups, a condition never before seen in “normal” heparin.
To enrich/purify the contaminant, several methods were attempted: One group attempted enrichment by degrading the heparin. Another used an alcohol based precipitation, while the third used chromatography, based on charge differences.
These purified samples were subjected to a battery of NMR techniques: HSQC (Heterocyclic Singular Quantum Correlation), COSY (COrrelation SpectroscopY), TOCSY (TOtal Correlation SpectroscopY), ROSEY (Rotating-frame OverhauSer Effect spectroscopY), and HMBC (Heteronuclear Multiple Bond Correlation). Basically, combinations of these techniques “map” the entire structure of the molecules, showing all links.
The bottom line was the discovery of a “heparin-like” molecule, made of 2,3-O-sulfoglucuronic acid and 4,6-O-sulfo-N-acetylgalactosamine, with a β-1,3-linkage between the two sugars in the disaccharide and a β-1,4-linkage between adjacent disaccharide units. The molecule was named oversulfated chondroitin sulfate (OSCS).
The molecule is not a natural product and was determined to have been synthesized and introduced into the heparin. The question still remained whether this was what caused the reactions in patients. Since similar compounds (e.g., the drug Arteparon, an arthritis medicine manufactured by a German company and which was removed from market after causing adverse reactions in patients) were found, it appeared that one of these might be the culprit. Another team of researchers from FDA, Harvard, MIT and VPI were assembled to determine the toxicity of the newly found compound.
The material was tested, ironically, on pigs and was found to cause the symptoms reported by the patients/doctors using the questionable heparin. Apparently, the chemical does not affect rodents and was not seen in routine screenings. The findings were published in April, two days after Chinese regulators held a press conference stating that OSCS was not the cause of the adverse reactions.
This begs any number of questions:
- Since the country of origin will not accept responsibility, we need some government agency in the US to do so. Who?
- And, more importantly, can any agency do so under current law?
- The amount of work needed for just one compound makes it obvious that this could be an expensive proposition. Who will pay?
- The work will require a large number of labs and scientists. Who will perform the work and where will it be done?
- Who will coordinate the work?
- Will the results be open to all pharma companies or only ones with money to contribute to the research?
- Can we “share” the expense with suppliers or will the customers (and taxpayers) have to “eat” the cost in higher drug prices and taxes?
- Should the industry reconsider the “benefits” of outsourcing to a country with a history of ignoring patents and copyrights?
- Of course, we also have to ask which products (only APIs or all excipients) will be tested and in which order?
- Who will determine the risk factors, if there wasn’t preliminary work performed? [We may not know a candidate for testing until a spate of people get sick or die!
- How many inspectors will the FDA need to do all this? When will they be hired? Trained? Deployed? What authority will they have?