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Scientists have used stable isotope analysis for decades, employing isotope ratio mass spectrometry to trace the isotopic fingerprints of natural materials. Within the past few years, pharmaceutical manufacturers and regulators have also begun to test the technique, which is relatively inexpensive and extremely precise, to authenticate pharmaceutical ingredients and products (Figures 1-3; Refs. 1-5).
In addition, isotopic analysis can also provide information about the manufacturing process, during which raw materials are manufactured into intermediates, active pharmaceutical ingredients (APIs) and, finally, into drug products. Stable-isotopic values are affected by the fractionation that occurs during the manufacturing process (Figure 4). The measurement of variables such as reaction type and rate, and separation processes such as recrystallization can be used to monitor reactions in various steps of Process Analytical Chemistry (PAC).
Recently, Molecular Isotope Technologies LLC (Niantic, Conn.) conducted joint studies with FDAs Division of Pharmaceutical Analysis using isotopic analysis to study the API, Naproxen [6,7]. Now, Molecular Isotope Technologies and Johnson & Johnson (J&J) are exploring the techniques potential in helping identify counterfeit products based on their processes, or to provide information in process patent infringement cases .
In this article, we discuss how isotopic analysis can be used to track the isotopic fractionation that occurs in synthetic pharmaceutical manufacturing processes. We also present and discuss preliminary results of J&J studies using isotopic analysis to evaluate the antiepileptic drug, Topiramate. So far, these results suggest that this analytical tool could be a practical, cost-effective way to analyze and control pharmaceutical manufacturing processes.
Fundamentals of isotopic analysis
To very briefly review the basics, stable isotopes exist naturally as mass variants of chemical elements. Carbon-13, for instance, consisting of seven neutrons and six protons, accounts for approximately 1.1 percent of all carbon in nature; carbon-12 accounts for approximately 98.9 percent. The variation in the 13C/12C ratiosand other isotopic ratios of pharmaceutical componentscan be used to trace the isotopic provenance or fingerprint of APIs or drug products via isotope-ratio mass spectrometry. Figure 1 is a schematic diagram of an Elemental Analyzer/Mass Spectrometer. In an EAMS, samples are dropped from a carousel to a high-temperature oven where they are combusted to small molecules such as CO2 or N2, whose isotope ratios are subsequently measured on an isotope ratio mass spectrometer.
When both the sources of starting materials and of the manufacturing process are fixed, stable-isotopic values for the products will be predictably constant and provide an identifiable and reproducible fingerprint. Over the last six years, authentication of pharmaceutical APIs and drug products, based on their isotopic compositions, has been shown to be a highly specific means of identifying the manufacturers and individual batches of products [1-5]. Both pharmaceutical manufacturers and regulatory agencies are interested in the technology as a forensic layer of product security.
However, FDA-DPAs Naproxen studies show that natural stable-isotopic ratios can also be measured to identify pharmaceutical material sources and determine the processes by which APIs are made (Figure 2). In 2002, J&J decided to test the techniques effectiveness in distinguishing between 53 samples of Topiramate that were made by three different synthetic pathways [6,7]. The initial goal was to determine whether the technique could be used as a tool in process patent infringement cases.
The observed isotopic results for the Topiramate samples can be grouped into a relatively small number of clusters consistent with synthetic fractionation. Three major isotopic-fractionating processes include:
- Synthetic reaction pathways (reaction mechanisms determining batch-to-batch isotopic variations via synthetic fractionation)
- Fractional crystallization
- Size fractionation (sieving, particle sorting).
Isotopic fractionation between light and heavy isotopes occurs when chemical reactions do not proceed to completion, or when multiple products are formed, and those isotopes are unevenly distributed among the reactants and products. In principle, the isotopic compositions of chemical products can be predicted from the isotopic compositions of the starting materials together with knowledge of the fractionations. However, fractionations can be quantitatively predicted only when complete mass balances are available and when the kinetic and equilibrium isotope effects associated with all relevant chemical reactions are accurately known .
Synthetic isotope fractionations are potential quantitative process monitors that could be used to integrate specific reaction variables that contribute to the isotopic composition of the synthetic product. In a given process for which the isotopic compositions of the reactants are known and the synthetic-isotopic fractionation has previously been determined, the isotopic composition of the product has a predictable isotopic value. If the observed value is not as predicted, then something in the synthetic process has varied. That may have been the reaction rate as modulated by factors such as pressure, temperature, reagent abundance, etc. Isotopic composition integrates such reaction variables and therefore can be used to monitor them.
Preliminary studies seek to differentiate the effects of raw materials from synthetic pathways in the synthesis of pharmaceutical intermediates and, ultimately, the synthetic fractionation of either partial or total synthetic pathways. When those factors are quantified, they should be reproducible, so that they present a means by which to monitor the consistency of synthetic processes. We have just begun this endeavor, but decades of isotopic experience in the earth sciences provide a solid background for such research.
Encouraging early results
Although the isotopic compositions of the starting materials used to produce the archived Topiramate samples were not controlled, the isotopic results were graphically grouped or clustered depending on the synthetic pathway used. The clustering of the data in Figure 3 into a small number of groups (rather than a random distribution) is consistent with either or both a limited number of synthetic pathways and/or a limited number of raw-material sources. In ongoing research, we are controllingand thus, developing a quantitative understanding ofthe specific isotopic contributions of each the starting materials and the synthetic processes to the products observed isotopic composition.
While the observed clustering may be related to only the different synthetic pathways used, effects due to the varying isotopic composition of the reagents and starting materials may also have contributed. Because the samples had been produced in a variety of different laboratories and manufacturing plants throughout the world, it was not possible to trace the sources of suppliers of the various raw materials, reagents, and exact reaction conditions used during manufacture.
At the present stage of development, stable-isotopic analyses are being performed offline at a separate analytical facility. However, we envision at-line isotopic analyses to become the preferred mode of application for synthetic-isotope analyses. At-line massspectroscopic analyses are already underway in the pharmaceutical industry . In fact, in addition to mass spectrometer manufacturers that produce laboratory instruments (Thermo Electron, San Jose, Calif., and GV Instruments, Manchester, U.K.), at least one firm, Monitor Instruments (Cheswick, Pa.), has already developed compact, on-line cycloid isotope-ratio mass spectrometry for the biochemical and geochemical industries.
At-line isotopic measurements can easily be performed on samples removed from the reaction stream. To our knowledge, there are presently no on-line methods for isotope-ratio measurements for solids such as pharmaceutical materials, although there are some for gases. However, given the existing multi-sample carousel systems for isotope analysis, only an at-line sampling system would be necessary to acquire nearly real-time samples and analyses.
The prospect for stable-isotopic analysis in PAT is generally promising since the ideas of synthetic-isotope fractionation described here have been developed and well accepted in the earth-science isotope community for decades. The dynamic range (= observed range/1 precision) of isotope-ratio mass spectrometric measurements for pharmaceutical materials is typically high (~50-100), indicating a marked utility for quantifying authentication of both products and processes.
Since 1999, pharmaceutical-isotope analysis has progressed from stable-isotopic authentication, largely observational work, of pharmaceutical products to the early stages of stable-isotopic analysis in PAC. The basic scientific principles for understanding stable-isotopic fractionation as natural process monitors have been in place for decades. We are just now beginning to evaluate them for industrial application. The FDA PAT guidance  and the cGMP initiative  encourage the implementation of new technologies. Assuming that we find adequate dynamic ranges in process measurements of interest, it is reasonable to expect that stable-isotope mass spectrometry may become one of the analytical technologies used in PAT.
Acknowledgements: The authors thank John M. Hayes for important contributions to this paper on synthetic-isotope fractionation.
About the Authors
Dr. John P. Jasper is the Chief Scientific Officer of Molecular Isotope Technologies LLC (www.MolecularIsotopes.com). He is an analytical organic and stable isotope chemist who uses bulk- and compound-specific isotopic approaches to determine the sources of natural and synthetic organic matter, particularly drug substances and drug products. He has a B.A. in Geophysical Sciences and Biological Sciences from the University of Chicago, and a Ph.D. in marine organic and isotopic chemistry from the M.I.T./Woods Hole Oceanographic Institution Joint Program in Chemical Oceanography.
Dr. Robbe C. Lyon is the Deputy Director of the Division of Product Quality Research, FDA CDER. He is the Process Analytical Technology (PAT) Research Team Leader. He earned a B.S. and M.S. in Physics and a Ph.D. in Biochemistry from Washington State University. He is a member of the PQRI Drug Substance Technical Committee, the FDA Drug Substance Technical Committee and was a member of the FDA Commissioners Counterfeit Drug Task Force.
Dr. Larry E. Weaner is a Senior Research Fellow at Johnson & Johnson in Spring House, Pa. He is responsible for the Radiosynthesis Group that provides radiolabeled compounds for use in drug discovery and development projects. He earned his Ph.D. in chemistry at Drexel University.
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