Skip to main content

NMR in the Characterization of Complex Mixture Drugs

  • Chapter
  • First Online:
The Science and Regulations of Naturally Derived Complex Drugs

Abstract

The number of complex drug substances, including biologics, present in different pharmacopoeias has increased in recent decades. These drugs are distinct from chemically synthesized pharmaceutical products, since they do not represent a single entity, but a complex combination of substances. They are usually produced by biotechnological methods, involving extraction from natural sources including human, animal, and plant tissue, microorganisms or, they can be prepared chemically (synthetically, semi-synthetically, and/or chemo-enzymatically). Among the different analytical approaches developed to assess the structural characteristics of complex drugs , NMR is one of the leading techniques. In the present chapter, examples of the latest techniques applied to the characterization of complex drugs are described.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Alam TM, Alam MK. Chemometric analysis of NMR spectroscopy data: a review. Annu Rep NMR Spectro. 2004;54:41–80.

    Google Scholar 

  2. Alban S, Lühn S, Schiemann S, Beyer T, Norwig J, Schilling C, Rädler O, Wolf B, Matz M, Baumann K, Holzgrabe U. Comparison of established and novel purity tests for the quality control of heparin by means of a set of 177 heparin samples. Anal Bioanal Chem. 2011;399:605–20.

    Article  CAS  Google Scholar 

  3. Amezcua CA, Szabo CM. Assessment of higher order comparability in therapeutic proteins using nuclear magnetic resonance spectroscopy. J Pharm Sci. 2013;102:1724–33.

    Article  CAS  Google Scholar 

  4. Anderson J, Ganguly T, Capila I, Bell C, Prescott J, Lansing J, Glajch J. Demonstration of equivalence between a generic (M356) and brand Copaxone® (glatiramer acetate injection) (P1.145). Neurology. 2015;84(14) Suppl P1:145.

    Google Scholar 

  5. Arnaud CH. High-resolution NMR structure of Parkinson’s disease fibril solved. Chem Eng News. 2016;94:7.

    Google Scholar 

  6. Aubin Y, Gingras G, Sauve S. Assessment of the three dimensional structure of recombinant protein therapeutics by NMR fingerprinting. Demonstration of human recombinant human granulocyte macrophage-colony stimulation factor. Anal Chem. 2008;80:2623–7.

    Article  CAS  PubMed  Google Scholar 

  7. Awotwe-Otoo D, Agarabi C, Keire DA, Lee S, Raw A, Yu L, Habib MJ, Khan MA, Shah RB. Physicochemical characterization of complex drug substances: Evaluation of structural similarities and differences of protamine sulfate from various sources. AAPS J. 2012;14:619–26.

    Article  CAS  PubMed  Google Scholar 

  8. Barrowcliffe TW. History of heparin. In Lever R, Mulloy B, Page PC, Barrowcliffe TW, editors. Heparin—a century of progress, von Heparin—a century of progress. Berlin, Heidelberg: Springer; 2012. p. 3–22.

    Google Scholar 

  9. Beyer T, Matz M, Brinz D, Rädler O, Wolf B, Norwig J, Baumann K, Alban S, Holzgrabe U. Composition of OSCS-contaminated heparin occurring in 2008 in batches on the German market. Eur J Pharm Sci. 2010;40:297–304.

    Article  CAS  PubMed  Google Scholar 

  10. Biljan I, Ilc G, Giachin G, Legname G, Plavec J. NMR structural studies of human cellular prion proteins. Curr Top Med Chem. 2013;13:2407–18.

    Article  CAS  Google Scholar 

  11. Breton RC, Reynolds WF. Using NMR to identify and characterize natural products. Nat Prod Rep. 2013;30:501–24.

    Article  CAS  Google Scholar 

  12. Casu B, Guerrini M, Naggi A, Torri G, De Ambrosi L, Boveri G, Gonella S, Cedro G, Ferro AL, Lanzarotti E, Paternò M, Attolini M. Characterization of sulfation patterns of beef and pig mucosal heparins by nuclear magnetic resonance spectroscopy. Arzneim Forsch Drug Res. 1996;46:472–7.

    CAS  Google Scholar 

  13. Casu B, Naggi A, Torri G. Revisiting the structure of heparin. Carbohydr Res. 2015;403:60–8.

    Article  CAS  Google Scholar 

  14. Casu B, Oreste P, Torri G, Zoppetti G, Choay J, Lormeau LC, Petitou M, Sinäy P. The structure of heparin oligosaccharide fragments with high anti-(factor Xa) activity containing the minimal antithrombin III-binding sequence. Chemical and 13C nuclear-magnetic-resonance studies. Biochem J. 1981;197:599–609.

    Article  CAS  PubMed  Google Scholar 

  15. Chen K, Freedberg DI, Keire DA. NMR profiling of biomolecules at natural abundance using 2D H-1-N-15 and H-1-C-13 multiplicity-separated (MS) HSQC spectra. J Magn Reson. 2015;251:65–70.

    Article  CAS  Google Scholar 

  16. Chen K, Long DS, Lute SC, Levy MJ, Brorson KA, Keire DA. Simple NMR methods for evaluating higher order structures of monoclonal antibody therapeutics with quinary structure. J Pharm Biomed Anal. 2016;128:398–407.

    Article  CAS  PubMed  Google Scholar 

  17. Choay J, Petitou M, Lormeau JC, Sinay P, Casu B, Gatti G. Structure-activity relationship in heparin: a synthetic pentasaccharide with high affinity for antithrombin III and eliciting high anti-factor Xa activity. Biochem Biophys Res Com. 1983;116:492–9.

    Article  CAS  Google Scholar 

  18. Daus S, Petzold-Welcke K, Kotteritzsch M, Baumgaertel A, Schubert US, Heinze T. Homogeneous sulfation of xylan from different sources. Macromol Mater Eng. 2011;296:551–61.

    Article  CAS  Google Scholar 

  19. De Ferra L, Naggi A, Zenoni M. EU Patent WO2014114723 A1. 2014.

    Google Scholar 

  20. Degenhardt M, Benend H, Wätzig H. Quality control of pentosane polysulfate by capillary zone electrophoresis using indirect detection. J Chromatogr A. 1998;817:297–306.

    Article  CAS  Google Scholar 

  21. Ebringerova A, Heinze T. Naturally occurring xylans structures, isolation procedures and properties. Macromol Rapid Commun. 2000;21:542–56.

    Article  CAS  Google Scholar 

  22. FDA. Pentosan polysulfate sodium. US Food and Drug Administration; 2013. http://www.accessdata.fda.gov/scripts/cder/drugsatfda/index.cfm.

  23. FDA. Response to heparin contamination helped protect public health; controls that were needed for working with external entities were recently added. Washington: US Government Accountability Office; 2010.

    Google Scholar 

  24. Ferro DR, Provasoli A, Ragazzi M, Torri G, Casu B, Gatti G, Jacquinet JC, Sinaÿ P, Petitou M, Choay JJ. Evidence for conformational equilibrium of the sulfated L-iduronate residue in heparin and in synthetic heparin mono- and oligo-saccharides: NMR and force-field studies. J Am Chem Soc. 1986;108:6773–8.

    Article  CAS  Google Scholar 

  25. Fu L, Li G, Yang B, Onishi A, Li L, Sun P, Zhang F, Linhardt RJ. Structural characterization of pharmaceutical heparins prepared from different animal tissues. J Pharm Sci. 2013;102:447–57.

    Article  Google Scholar 

  26. Gatti G, Casu B, Hamer AS, Perlin AS. Studies on the conformation of Heparin by 1H and 13C NMR spectroscopy. Macromolecules. 1979;12:1001–7.

    Article  CAS  Google Scholar 

  27. Ghasriani H, Hodgson DJ, Brinson RG, McEwen I, Buhse LF, Kozlowski S, Marino JP, Aubin Y, Keire DA. Precision and robustness of 2D-NMR for structure assessment of filgrastim biosimilars. Nat Biotechnol. 2016;34:139–41.

    Article  CAS  PubMed  Google Scholar 

  28. Gilard V, Trefi S, Balayssac S, Delsuc MA, Gostan T, Malet-Martino M, Martino R, Prigent Y, Taulelle F. DOSY NMR for drug analysis. In von Wawer I, Dihel I, Holtzgrabe U, editors. NMR spectroscopy in pharmaceutical analysis. Oxford: Elsevier; 2008. p. 269–87.

    Chapter  Google Scholar 

  29. Giraudeau P. Quantitative 2D liquid-state NMR. Magn Reson Chem. 2014:259–72.

    Article  CAS  Google Scholar 

  30. Gray E, Mulloy B, Barrowcliffe TW. Heparin and low-molecular-weight heparin. Thromb Haemost. 2008;99:807–18.

    Article  CAS  Google Scholar 

  31. Gronwald W, Klein MS, Kaspar H, Fagerer SR, Nurnberger N, Dettmer K, Bertsch T, Oefner PJ. Urinary metabolite quantification employing 2D NMR spectroscopy. Anal Chem. 2008;80:9288–97.

    Article  CAS  PubMed  Google Scholar 

  32. Gucinski AC, Boyne MT, Keire DA. Modern analytics for naturally derived complex drug substances: NMR and MS tests for protamine sulfate from chum salmon. Anal Bioanal Chem. 2015;407:749–59.

    Article  CAS  PubMed  Google Scholar 

  33. Guerrini M, Beccati D, Shriver Z, Naggi A, Viswanathan K, Bisio A, Capila I, Lansing JC, Guglieri S, Fraser B, Al-Hakim A, Gunay NS, Zhang Z, Robinson L, Buhse L, Nasr M, Woodcock J, Langer R, Venkataraman G, Linhardt RJ, Casu B, Torri G, Sasisekharan R. Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events. Nat Biotechnol. 2008;26:669–75.

    Article  CAS  PubMed  Google Scholar 

  34. Guerrini M, Bisio A. Low-molecular-weight Heparins: differential characterization/physical characterization. In von Mulloy L, Page B, Lever CP, editors. Heparin—a century of progress. Berlin: Springer; 2012. p. 127–57.

    Google Scholar 

  35. Guerrini M, Bisio A, Torri G. Combined quantitative 1H and 13C nuclear magnetic resonance spectroscopy for characterization of Heparin preparations. Sem Thromb Hemost. 2001;5:473–82.

    Article  Google Scholar 

  36. Guerrini M, Mourier PAJ, Torri G, Viskov C. Antithrombin-binding oligosaccharides: structural diversities. Glycoconj J. 2014;31:409–16.

    Article  CAS  PubMed  Google Scholar 

  37. Guerrini M, Naggi A, Guglieri S, Santarsiero R, Torri G. Complex glycosaminoglycans: profiling substitution patterns by two dimensional NMR spectroscopy. Anal Biochem. 2005;337:35–47.

    Article  CAS  PubMed  Google Scholar 

  38. Guerrini M, Rudd TR, Mauri L, Macchi E, Fareed J, Yates EA, Naggi A, Torri G. Differentiation of generic enoxaparins marketed in the United States by employing NMR and multivariate analysis. Anal Chem. 2015;87:8275–83.

    Article  CAS  Google Scholar 

  39. Guerrini M, Guglieri S, Naggi A, Torri G. LMWHs: structural differentiation by bidimensional NMR spectroscopy. Seminars Thromb Hemos. 2007;33:478–87.

    Article  CAS  Google Scholar 

  40. Heikkinen S, Toikka MM, Karhunen PT, Kilpeläinen A. Quantitative 2D HSQC (Q-HSQC) via suppression of J-dependence of polarization transfer in NMR spectroscopy: application to wood lignin. J Am Chem Soc. 2003;125:4362–7.

    Article  CAS  PubMed  Google Scholar 

  41. Holzgrabe U, Wawer I, Diehl B. NMR spectroscopy in pharmaceutical analysis. Oxford; 2008 (Holzgrabe U, Wawer I, Diehl B).

    Google Scholar 

  42. Holzgrabe U. Quantitative NMR spectroscopy in pharmaceutical applications. Prog Nucl Magn Reson Spectrosc. 2010;57:229–40.

    Article  CAS  PubMed  Google Scholar 

  43. Hricovini M, Guerrini M, Bisio A, Torri G, Petitou M, Casu B. Conformation of heparin pentasaccharide bound to antithrombin. Biochem J. 2001;359:265–72.

    Article  CAS  PubMed  Google Scholar 

  44. Hwang P, Auclair B, Beechinor D, Diment M, Einarson TR. Efficacy of pentosan polysulfate in the treatment of interstitial cystitis: a meta-analysis. Urology. 1997;50:39–43.

    Article  CAS  PubMed  Google Scholar 

  45. Johnson CS Jr. Diffusion ordered nuclear magnetic resonance spectroscopy: principles and applications. Prog Nucl Magn Reson Spectrosc. 1999;34:203–56.

    Article  CAS  Google Scholar 

  46. Jolliffe IT. Principal component analysis. 2nd ed (Bickel P, Diggle P, Fienberg S, Krickeberg K, Olkin I, Wermuth N, Zeger S). Springer; 2002.

    Google Scholar 

  47. Keire DA, Buhse LF, Al-Hakim A. Characterization of currently marketed heparin products: composition analysis by 2D-NMR. Anal Methods. 2013;5:2984–94.

    Article  CAS  Google Scholar 

  48. Keire DA, Mulloy B, Chase C, Al-Hakim A, Cairatti D, Gray E, Hogwood J, Morris T, Mourão P, Carvalho Soares MDS, Szajek A. Diversifying the global Heparin supply chain: reintroduction of Bovine Heparin in the United States? Pharm Technol. 2015;39(11).

    Google Scholar 

  49. Lanthier M, Behrman R, Nardinelli C. Economic issues with follow-on protein products. Nat Rev Drug Discov. 2008;7:733–7.

    Article  CAS  PubMed  Google Scholar 

  50. Le Guennec A, Tea I, Antheaume I, Martineau E, Charrier B, Pathan M, Akoka S, Giraudeau P. Fast determination of absolute metabolite concentrations by spatially-encoded 2D NMR: application to breast cancer cell extracts. Anal Chem. 2012;84:10831–7.

    Article  Google Scholar 

  51. Lee S, Raw A, Yu L, Lionberger R, Ya N, Verthelyi D, Rosenberg A, Kozlowski S, Webber K, Woodcock J. Scientific considerations in the review and approval of generic enoxaparin in the United States. Nat Biotechnol. 2013;31:220–6.

    Article  CAS  PubMed  Google Scholar 

  52. Levy MJ, Gucinski AC, Sommers CD, Ghasriani H, Wang B, Keire DA, Boyne MT. Analytical techniques and bioactivity assays to compare the structure and function of filgrastim (granulocyte-colonystimulating factor) therapeutics from different manufacturers. Anal Bioanal Chem. 2014;406:6559–67.

    Article  CAS  Google Scholar 

  53. Lewis IA, Schommer SC, Hodis B, Robb KA, Tonelli M, Westler W, Sussman M, Markley JL. Method for determining molar concentrations of metabolites in complex solutions from two-dimensional 1H-13C NMR spectra. Anal Chem. 2007;79:9385–90.

    Article  CAS  PubMed  Google Scholar 

  54. Mauri L, Boccardi G, Torri G, Karfunkle M, Macchi E, Muzi L, Keire DA, Guerrini M. Qualification of HSQC methods for quantitative composition of heparin and low molecular weight heparins. J Pharm Biomed Anal;2016: submitted.

    Google Scholar 

  55. Mourier PAJ, Guichard O, Herman F, Viskov C. Isolation of a pure octadecasaccharide with antithrombin activity from an ultra-low-molecular-weight heparin. Anal Biochem. 2014;453:7–15.

    Article  CAS  Google Scholar 

  56. Naggi A, Gardini C, Pedrinola G, Mauri L, Urso E, Alekseeva A, Casu B, Cassinelli G, Guerrini M, Iacomini M, Baigorria V, Torri G. Structural peculiarity and antithrombin binding region profile of mucosal bovine and porcine heparins. J Pharm Biomed Anal. 2015;118:52–63.

    Article  Google Scholar 

  57. Parson IC, Lansing J, Bosques CY, Beccati D. United States Patent US 2010/0188084; 2010.

    Google Scholar 

  58. Pauli GF, Gödecke T, Jaki BU, Lankin DC. Quantitative 1H NMR. Development and potential of an analytical methods: an update. J Nat Prod. 2012;75:834–51.

    Article  CAS  PubMed  Google Scholar 

  59. Perlin AS, Mazurek M, Jaques LB, Kavanak LW. Proton magnetic resonance spectral study of heparin. Carbohydr Res. 1968;7:369–79.

    Article  CAS  Google Scholar 

  60. Rogstad S, Pang E, Sommers C, Hu M, Jiang X, Keire DA, Boyne MT. Modern analytics for synthetically derived complex drug substances: NMR, AFFF–MALS, and MS tests for glatiramer acetate. Anal Bioanal Chem. 2015;407:8647–59.

    Article  CAS  Google Scholar 

  61. Rudd TR, Gaudesi D, Lima MA, Skidmore MA, Mulloy B, Torri G, Nader HB, Guerrini M, Yates EA. High-sensitivity visualisation of contaminants in heparin samples by spectral filtering of 1H NMR spectra. Analyst. 2011;136:1390–8.a Rudd TR, Gaudesi D, Skidmore MA, Ferro M, Guerrini M, Mulloy B, Torri G, Yates EA. Construction and use of a library of bona fide heparins employing 1H NMR and multivariate analysis. Analyst. 2011:1380–9.b

    Article  CAS  Google Scholar 

  62. Rudd TR, Macchi E, Gardini C, Muzi L, Guerrini M, Yates EA, Torri G. How to find a needle (or anything else) in a haystack: two-dimensional correlation spectroscopy-filtering with iterative random sampling applied to pharmaceutical heparin. Anal Chem. 2012;84:6841–7.

    Article  CAS  Google Scholar 

  63. Rudd TR, Macchi E, Muzi L, Ferro M, Gaudesi D, Torri G, Casu B, Guerrini M, Yates EA. Unravelling structural information from complex mixtures utilizing correlation spectroscopy applied to HSQC spectra. Anal Chem. 2013;85:7487–93.

    Article  CAS  Google Scholar 

  64. Rudd TR, Skidmore MA, Guimond SE, Cosentino C, Torri G, Fernig DG, Lauder RM, Guerrini M, Yates EA. Glycosaminoglycan origin and structure revealed by multivariate analysis of NMR and CD spectra. Glycobiology. 2009;19:62–7.

    Article  Google Scholar 

  65. Ruiz-Calero V, Saurina J, Galceran MT, Hernandez-Cassou S, Puignou L. Estimation of the composition of heparin mixtures from various origins using proton nuclear magnetic resonance and multivariate calibration methods. Anal Bioanal Chem. 2002;373:259–65.

    Article  CAS  Google Scholar 

  66. Ruiz-Calero V, Saurina J, Galceran MT, Hernandez-Cassou S, Puignou L. Potentiality of proton nuclear magnetic resonance and multivariate calibration methods for the determination of dermatan sulfate contamination in heparin samples. Analyst. 2000;125:933–8.

    Article  CAS  Google Scholar 

  67. Sarwar MI, Hussain MS, Leghari AR. Heparin can be isolated and purified from bovine intestine by different techniques. Int J Pharm Sci Invent. 2013;2:21–5.

    CAS  Google Scholar 

  68. Sassaki GL, Guerrini M, Serrato RV, Santana Filho AP, Carlotto J, Simas-Tosin F, Cipriani TR, Iacomini MG. Torri G, Gorin PAJ. Monosaccharide composition of glycans based on Q-HSQC NMR. Carbohydr Polym. 2013;104:34–41.

    Article  CAS  Google Scholar 

  69. Schellekens H, Klinger E, Mühlebach S, Brin JF, Storm G, Crommelin DJ. The therapeutic equivalence of complex drugs. Toxicol Pharm. 2011;59:176–83.

    CAS  Google Scholar 

  70. Schrempf W, Ziemssen T. Glatiramer acetate: mechanisms of action in multiple sclerosis. Autoimmun Rev. 2007;6:469–75.

    Article  CAS  Google Scholar 

  71. Stewart KL, Hughes E, Yates EA, Akien GR, Huang T, Lima MA, Rudd TR, Guerrini M, Hung T, Radford SE, Middleton DA. Atomic details of the interactions of glycosaminoglycans with amyloid–β fibrils. J Am Chem Soc. 2016;138:8328–31.

    Article  CAS  Google Scholar 

  72. Szajek AY, Chess E, Johansen K, Gratzl G, Gray E, Keire D, Linhardt RJ, Liu J, Morris T, Mulloy B, Nasr M, Shriver Z, Torralba P, Viskov C, Williams R, Woodcock J, Workman W, Al-Hakim A. The US regulatory and pharmacopeia response to the global heparin contamination crisis. Nat Biotechnol. 2016;34:625–30.

    Article  CAS  Google Scholar 

  73. Teitelbaum D, Meshorer A, Hirshfeld T, Arnon R, Sela M. Supression of experimental allergic encephalomyelitis by a synthetic polypeptide. Eur J Immunol. 1971;1:242–8.

    Article  CAS  Google Scholar 

  74. Teitelbaum D, Meshorer A, Hirshfeld T, Arnon R, Sela M. USA Patent 3849550; 1974.

    Google Scholar 

  75. Viskov C, Elli S, Urso E, Gaudesi D, Mourier P, Herman F, Boudier C, Casu B, Torri G, Guerrini M. Heparin Dodecasaccharide containing two antithrombin-binding pentasaccharides. Structural features and biological properties. J Biol Chem. 2013;288:25895–907.

    Article  CAS  PubMed  Google Scholar 

  76. Walsh G. Biopharmaceutical benchmarks. Nat Biotech. 2018;36(12):1136.

    Google Scholar 

  77. Wolfrom ML, Rice FAH. The uronic acid component of Heparin. J Am Chem Soc. 1946;68:532.

    Article  CAS  PubMed  Google Scholar 

  78. Wuthrich K. NMR in structural biology. Singapore: K. Wuthrich; 1995.

    Book  Google Scholar 

  79. Xiao Z, Tappen BR, Ly M, Zhao W, Canova LP, Guan H, Linhardt RJ. Heparin mapping using heparin lyases and the generation of a novel low molecular weight heparin. J Med Chem. 2011;54:603–10.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Marco Guerrini .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2019 American Association of Pharmaceutical Scientists

About this chapter

Check for updates. Verify currency and authenticity via CrossMark

Cite this chapter

Guerrini, M., Rudd, T.R., Yates, E.A. (2019). NMR in the Characterization of Complex Mixture Drugs. In: Sasisekharan, R., Lee, S., Rosenberg, A., Walker, L. (eds) The Science and Regulations of Naturally Derived Complex Drugs. AAPS Advances in the Pharmaceutical Sciences Series, vol 32. Springer, Cham. https://doi.org/10.1007/978-3-030-11751-1_7

Download citation

Publish with us

Policies and ethics