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  • Review Article
  • Published:

Analytical tools for characterizing biopharmaceuticals and the implications for biosimilars

Nature Reviews Drug Discovery volume 11, pages 527–540 (2012)Cite this article

Subjects

Key Points

  • Therapeutic biologics such as monoclonal antibodies possess substantial structural complexities in comparison with small-molecule drugs.

  • Biologics represent the fastest growing segment of the pharmaceutical market; to gain regulatory approval of these agents, developers of these products are required to perform in-depth characterization to ensure batch-to-batch consistency. In addition to new products developed by innovators, the market will also see the introduction of follow-on biologics (also known as biosimilars), and regulatory agencies (including the US Food and Drug Administration in 2012) have been prompted to develop specific guidelines for this class of drugs.

  • The regulatory pathways for follow-on versions of complex biologics require the comparison of a reference (innovator) product to the biosimilar version. Analytical technologies provide vital product characterization data that can help establish the degree of comparability and similarity. Such data are likely to have impact on the product approval process, including the scope of required clinical trials, the shelf life of the approved version and the requirements for post-market assessment of the product.

  • This Review discusses the current state of the art in the analytical technologies used to structurally characterize complex biologics, and also addresses properties of biologics that regulatory authorities have identified as being important in any development strategy for biosimilar versions.

  • Specific emphasis is placed on the analysis of post-translational protein modifications, three-dimensional protein structures, protein aggregates and subvisible particles that are present in formulated products.

Abstract

Biologics such as monoclonal antibodies are much more complex than small-molecule drugs, which raises challenging questions for the development and regulatory evaluation of follow-on versions of such biopharmaceutical products (also known as biosimilars) and their clinical use once patent protection for the pioneering biologic has expired. With the recent introduction of regulatory pathways for follow-on versions of complex biologics, the role of analytical technologies in comparing biosimilars with the corresponding reference product is attracting substantial interest in establishing the development requirements for biosimilars. Here, we discuss the current state of the art in analytical technologies to assess three characteristics of protein biopharmaceuticals that regulatory authorities have identified as being important in development strategies for biosimilars: post-translational modifications, three-dimensional structures and protein aggregation.

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Figure 1: Uses of analytical tools in the characterization of biopharmaceuticals.

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References

  1. Walsh, G. Biopharmaceutical benchmarks 2010. Nature Biotech. 28, 917–924 (2010). This comprehensive survey on the biopharmaceutical marketplace, carried out every 4 years, offers an overview of the key trends in the industry and new biopharmaceutical approvals.

    Article  CAS  Google Scholar 

  2. Lawrence, S. Billion dollar babies — biotech drugs as blockbusters. Nature Biotech. 25, 380–382 (2007).

    Article  CAS  Google Scholar 

  3. Erickson, B. E. Untangling biosimilars. Chem. Eng. News 88, 25–27 (2010).

    Google Scholar 

  4. Woodcock, J. et al. The FDA's assessment of follow-on protein products: a historical perspective. Nature Rev. Drug Discov. 6, 437–442 (2007).

    Article  Google Scholar 

  5. Kozlowski, S., Woodcock, J., Midthun, K. & Sherman, R. B. Developing the nation's biosimilars program. N. Engl. J. Med. 365, 385–388 (2011). References 4 and 5 are two papers that were written by regulators at the FDA; these two papers have summarized in a compact form the agency's historical perspective on biosimilars — a perspective that became mostly encapsulated by the draft guidelines that were later issued.

    Article  CAS  PubMed  Google Scholar 

  6. European Medicines Agency. Guideline on similar biological medicinal products containing biotechnology-derived proteins as an active substance: quality issues. EMA website [online], (2005).

  7. McCamish, M. & Woollett, G. Worldwide experience with biosimilar development. MAbs 3, 209–217 (2011). This is a detailed treatment on the opportunities, comparability, development requirements and product attributes of biosimilars, and includes perspectives on how these molecules have been dealt with in the European Union.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Dowlat, H. A. Perception & realities of clinical safety of biosimilars — EU & US perspectives: part 1. Regulatory Rapporteur 9, 20–25 (2012).

    Google Scholar 

  9. European Generic medicines Association. EGA Docket response: Docket No. FDA-2010-N-0477 EGA website [online], (2010).

  10. US Food and Drug Administration. Potential need for measurement standards to facilitate R&D of biologic drugs: statement of Steven Kozlowski, M.D. before the U.S. House of Representatives. FDA website [online], (2009).

  11. Lubiniecki, A. et al. Comparability assessments of process and product changes made during development of two different monoclonal antibodies. Biologicals 39, 9–22 (2011).

    Article  CAS  PubMed  Google Scholar 

  12. Skrlin, A. et al. Comparison of the physicochemical properties of a biosimilar filgrastim with those of reference filgrastim. Biologicals 38, 557–566 (2010).

    Article  CAS  PubMed  Google Scholar 

  13. Liu, C. et al. Assessment of the quality and structural integrity of a complex glycoprotein mixture following extraction from the formulated biopharmaceutical drug product. J. Pharm. Biomed. Anal. 54, 27–36 (2011).

    Article  CAS  PubMed  Google Scholar 

  14. Panjwani, N., Hodgson, D. J., Sauve, S. & Aubin, Y. Assessment of the effects of pH, formulation and deformulation on the conformation of interferon alpha-2 by NMR. J. Pharm. Sci. 99, 3334–3342 (2010).

    Article  CAS  PubMed  Google Scholar 

  15. Deechongkit, S., Aoki, K. H., Park, S. S. & Kerwin, B. A. Biophysical comparability of the same protein from different manufacturers: a case study using epoetin alfa from Epogen and Eprex. J. Pharm. Sci. 95, 1931–1943 (2006).

    Article  CAS  PubMed  Google Scholar 

  16. Heavner, G. A., Arakawa, T., Philo, J. S., Calmann, M. A. & Labrenz, S. Protein isolated from biopharmaceutical formulations cannot be used for comparative studies: follow-up to “a case study using epoetin Alfa from Epogen and EPREX”. J. Pharm. Sci. 96, 3214–3225 (2007).

    Article  CAS  PubMed  Google Scholar 

  17. Farley, A. R. & Link, A. J. Identification and quantification of protein posttranslational modifications. Methods Enzymol. 463, 725–763 (2009).

    Article  CAS  PubMed  Google Scholar 

  18. Walsh, C. T., Garneau-Tsodikova, S. & Gatto, G. J. Jr. Protein posttranslational modifications: the chemistry of proteome diversifications. Angew. Chem. Int. Ed. Engl. 44, 7342–7372 (2005).

    Article  CAS  PubMed  Google Scholar 

  19. Walsh, G. & Jefferis, R. Post-translational modifications in the context of therapeutic proteins. Nature Biotech. 24, 1241–1252 (2006). References 17–19 provide a comprehensive overview of protein PTMs; reference 19 also covers the impact that these modifications have on the structure–function relationships of therapeutic proteins, with particular emphasis on glycosylation.

    Article  CAS  Google Scholar 

  20. Buttel, I. C. et al. Taking immunogenicity assessment of therapeutic proteins to the next level. Biologicals 39, 100–109 (2011).

    Article  CAS  PubMed  Google Scholar 

  21. Baker, M. P., Reynolds, H. M., Lumicisi, B. & Bryson, C. J. Immunogenicity of protein therapeutics: the key causes, consequences and challenges. Self Nonself 1, 314–322 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  22. Singh, S. K. Impact of product-related factors on immunogenicity of biotherapeutics. J. Pharm. Sci. 100, 354–387 (2011).

    Article  CAS  PubMed  Google Scholar 

  23. Wen, D. et al. Discovery and investigation of misincorporation of serine at asparagine positions in recombinant proteins expressed in Chinese hamster ovary cells. J. Biol. Chem. 284, 32686–32694 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Schiestl, M. et al. Acceptable changes in quality attributes of glycosylated biopharmaceuticals. Nature Biotech. 29, 310–312 (2011).

    Article  CAS  Google Scholar 

  25. Han, X., Aslanian, A. & Yates, J. R. Mass spectrometry for proteomics. Curr. Opin. Chem. Biol. 12, 483–490 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Chen, G. & Pramanik, B. N. LC-MS for protein characterization: current capabilities and future trends. Expert Rev. Proteom. 5, 435–444 (2008).

    Article  CAS  Google Scholar 

  27. Jaisson, S. & Gillery, P. Evaluation of nonenzymatic posttranslational modification-derived products as biomarkers of molecular aging of proteins. Clin. Chem. 56, 1401–1412 (2010).

    Article  PubMed  Google Scholar 

  28. Morelle, W. & Michalski, J. C. Analysis of protein glycosylation by mass spectrometry. Nature Protoc. 2, 1585–1602 (2007).

    Article  CAS  Google Scholar 

  29. Chen, G. et al. Characterization of protein therapeutics by mass spectrometry: recent developments and future directions. Drug Discov. Today 16, 58–64 (2011).

    Article  CAS  PubMed  Google Scholar 

  30. An, H. J., Froehlich, J. W. & Lebrilla, C. B. Determination of glycosylation sites and site-specific heterogeneity in glycoproteins. Curr. Opin. Chem. Biol. 13, 421–426 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Zhang, Z., Pan, H. & Chen, X. Mass spectrometry for structural characterization of therapeutic antibodies. Mass Spectrom. Rev. 28, 147–176 (2009).

    Article  CAS  PubMed  Google Scholar 

  32. Witze, E. S., Old, W. M., Resing, K. A. & Ahn, N. G. Mapping protein post-translational modifications with mass spectrometry. Nature Methods 4, 798–806 (2007).

    Article  CAS  PubMed  Google Scholar 

  33. Srebalus Barnes, C. A. & Lim, A. Applications of mass spectrometry for the structural characterization of recombinant protein pharmaceuticals. Mass Spectrom. Rev. 26, 370–388 (2007).

    Article  CAS  PubMed  Google Scholar 

  34. Jiang, H., Wu, S. L., Karger, B. L. & Hancock, W. S. Characterization of the glycosylation occupancy and the active site in the follow-on protein therapeutic: TNK-tissue plasminogen activator. Anal. Chem. 82, 6154–6162 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Xie, H. et al. Rapid comparison of a candidate biosimilar to an innovator monoclonal antibody with advanced liquid chromatography and mass spectrometry technologies. MAbs 2, 379–394 (2010).

    Article  PubMed  Google Scholar 

  36. Yu, Y. Q. et al. Analysis of N-linked glycans from recombinant and human plasma derived coagulation factor IX using HILIC LC/FLR/QTof MS. Proceedings of the 58th ASMS Conference on Mass Spectrometry ThP 032 (23–27 May 2010; Salt Lake City, Utah, USA).

    Google Scholar 

  37. Kilgore, B. R., Lucka, A. W., Patel, R., Andrien, B. A. Jr & Dhume, S. T. Comparability and monitoring immunogenic N-linked oligosaccharides from recombinant monoclonal antibodies from two different cell lines using HPLC with fluorescence detection and mass spectrometry. Methods Mol. Biol. 446, 333–346 (2008).

    Article  CAS  PubMed  Google Scholar 

  38. Chung, C. H. et al. Cetuximab-induced anaphylaxis and IgE specific for galactose-α-1,3-galactose. N. Engl. J. Med. 358, 1109–1117 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Schoenecker, J. G., Hauck, R. K., Mercer, M. C., Parker, W. & Lawson, J. H. Exposure to topical bovine thrombin during surgery elicits a response against the xenogeneic carbohydrate galactose α1-3galactose. J. Clin. Immunol. 20, 434–444 (2000).

    Article  CAS  PubMed  Google Scholar 

  40. Hokke, C. H. et al. Sialylated carbohydrate chains of recombinant human glycoproteins expressed in Chinese hamster ovary cells contain traces of N-glycolylneuraminic acid. FEBS Lett. 275, 9–14 (1990).

    Article  CAS  PubMed  Google Scholar 

  41. Borys, M. C. et al. Effects of culture conditions on N-glycolylneuraminic acid (Neu5Gc) content of a recombinant fusion protein produced in CHO cells. Biotechnol. Bioeng. 105, 1048–1057 (2010).

    CAS  PubMed  Google Scholar 

  42. Ghaderi, D., Taylor, R. E., Padler-Karavani, V., Diaz, S. & Varki, A. Implications of the presence of N-glycolylneuraminic acid in recombinant therapeutic glycoproteins. Nature Biotech. 28, 863–867 (2010).

    Article  CAS  Google Scholar 

  43. Marino, K., Bones, J., Kattla, J. J. & Rudd, P. M. A systematic approach to protein glycosylation analysis: a path through the maze. Nature Chem. Biol. 6, 713–723 (2010).

    Article  CAS  Google Scholar 

  44. Valliere-Douglass, J. F. et al. Asparagine-linked oligosaccharides present on a non-consensus amino acid sequence in the CH1 domain of human antibodies. J. Biol. Chem. 284, 32493–32506 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Kellie, J. F. et al. The emerging process of top down mass spectrometry for protein analysis: biomarkers, protein-therapeutics, and achieving high throughput. Mol. Biosyst. 6, 1532–1539 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Borchers, C. H. et al. Combined top-down and bottom-up proteomics identifies a phosphorylation site in stem-loop-binding proteins that contributes to high-affinity RNA binding. Proc. Natl Acad. Sci. USA 103, 3094–3099 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Coon, J. J. Collisions or electrons? Protein sequence analysis in the 21st century. Anal. Chem. 81, 3208–3215 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Siuti, N. & Kelleher, N. L. Decoding protein modifications using top-down mass spectrometry. Nature Methods 4, 817–821 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Wu, S. L. et al. Mass spectrometric determination of disulfide linkages in recombinant therapeutic proteins using online LC-MS with electron-transfer dissociation. Anal. Chem. 81, 112–122 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Mamula, M. J. et al. Isoaspartyl post-translational modification triggers autoimmune responses to self-proteins. J. Biol. Chem. 274, 22321–22327 (1999).

    Article  CAS  PubMed  Google Scholar 

  51. Doyle, H. A., Gee, R. J. & Mamula, M. J. Altered immunogenicity of isoaspartate containing proteins. Autoimmunity 40, 131–137 (2007).

    Article  CAS  PubMed  Google Scholar 

  52. Chan, W. Y., Chan, T. W. & O'Connor, P. B. Electron transfer dissociation with supplemental activation to differentiate aspartic and isoaspartic residues in doubly charged peptide cations. J. Am. Soc. Mass Spectrom. 21, 1012–1015 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Mukherjee, R., Adhikary, L., Khedkar, A. & Iyer, H. Probing deamidation in therapeutic immunoglobulin gamma (IgG1) by 'bottom-up' mass spectrometry with electron transfer dissociation. Rapid Commun. Mass Spectrom. 24, 879–884 (2010).

    Article  CAS  PubMed  Google Scholar 

  54. Li, X., Lin, C. & O'Connor, P. B. Glutamine deamidation: differentiation of glutamic acid and γ-glutamic acid in peptides by electron capture dissociation. Anal. Chem. 82, 3606–3615 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Sargaeva, N. P., Lin, C. & O'Connor, P. B. Identification of aspartic and isoaspartic acid residues in amyloid β peptides, including Aβ1–42, using electron-ion reactions. Anal. Chem. 81, 9778–9786 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Yang, H., Fung, E. Y., Zubarev, A. R. & Zubarev, R. A. Toward proteome-scale identification and quantification of isoaspartyl residues in biological samples. J. Proteome Res. 8, 4615–4621 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Ni, W., Dai, S., Karger, B. L. & Zhou, Z. S. Analysis of isoaspartic acid by selective proteolysis with Asp-N and electron transfer dissociation mass spectrometry. Anal. Chem. 82, 7485–7491 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. An, H. J. & Lebrilla, C. B. Structure elucidation of native N- and O-linked glycans by tandem mass spectrometry (tutorial). Mass Spectrom. Rev. 30, 560–578 (2011).

    Article  CAS  PubMed  Google Scholar 

  59. Guerrini, M. et al. Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events. Nature Biotech. 26, 669–675 (2008).

    Article  CAS  Google Scholar 

  60. Lin, Y., Schiavo, S., Orjala, J., Vouros, P. & Kautz, R. Microscale LC-MS-NMR platform applied to the identification of active cyanobacterial metabolites. Anal. Chem. 80, 8045–8054 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Moffat, K. & Chait, B. T. Biophysical methods: doing more with less. Curr. Opin. Struct. Biol. 13, 535–537 (2003).

    Article  CAS  PubMed  Google Scholar 

  62. Denslow, N. D., Wingfield, P. T. & Rose, K. Overview of the characterization of recombinant proteins. Curr. Protoc. Protein Sci. Chapter 7, Unit 7.1 (2001).

  63. Price, N. C. Conformational issues in the characterization of proteins. Biotechnol. Appl. Biochem. 31, 29–40 (2000).

    Article  CAS  PubMed  Google Scholar 

  64. Kaltashov, I. A. et al. Advances and challenges in analytical characterization of biotechnology products: mass spectrometry-based approaches to study properties and behavior of protein therapeutics. Biotechnol. Adv. 30, 210–222 (2012).

    Article  CAS  PubMed  Google Scholar 

  65. Sorgel, F., Lerch, H. & Lauber, T. Physicochemical and biologic comparability of a biosimilar granulocyte colony-stimulating factor with its reference product. BioDrugs 24, 347–357 (2010).

    Article  PubMed  Google Scholar 

  66. Aubin, Y., Gingras, G. & Sauve, S. Assessment of the three-dimensional structure of recombinant protein therapeutics by NMR fingerprinting: demonstration on recombinant human granulocyte macrophage-colony stimulation factor. Anal. Chem. 80, 2623–2627 (2008).

    Article  CAS  PubMed  Google Scholar 

  67. Zuperl, S., Pristovsek, P., Menart, V., Gaberc-Porekar, V. & Novic, M. Chemometric approach in quantification of structural identity/similarity of proteins in biopharmaceuticals. J. Chem. Inf. Model. 47, 737–743 (2007).

    Article  CAS  PubMed  Google Scholar 

  68. Abeygunawardana, C., Williams, T. C., Sumner, J. S. & Hennessey, J. P. Jr. Development and validation of an NMR-based identity assay for bacterial polysaccharides. Anal. Biochem. 279, 226–240 (2000).

    Article  CAS  PubMed  Google Scholar 

  69. Freedberg, D. I. Using nuclear magnetic resonance spectroscopy to characterize biologicals. Dev. Biol. (Basel) 122, 77–83 (2005).

    CAS  Google Scholar 

  70. Lundblad, R. L. Approaches to the Conformational Analysis of Biopharmaceuticals (Chapman Hall/CRC Press, 2009).

    Book  Google Scholar 

  71. Jiskoot, W. & Crommelin, D. J. (eds) Methods for Structural Analysis of Protein Pharmaceuticals (AAPS Press, 2005).

    Google Scholar 

  72. Engen, J. R. Analysis of protein conformation and dynamics by hydrogen/deuterium exchange MS. Anal. Chem. 81, 7870–7875 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Takamoto, K. & Chance, M. R. Radiolytic protein footprinting with mass spectrometry to probe the structure of macromolecular complexes. Annu. Rev. Biophys. Biomol. Struct. 35, 251–276 (2006). References 72 and 73 summarize the main mass spectrometry-based techniques for assessing the higher-order structure of proteins.

    Article  CAS  PubMed  Google Scholar 

  74. Chalmers, M. J. et al. Probing protein ligand interactions by automated hydrogen/deuterium exchange mass spectrometry. Anal. Chem. 78, 1005–1014 (2006).

  75. Rand, K. D., Zehl, M., Jensen, O. N. & Jorgensen, T. J. Protein hydrogen exchange measured at single-residue resolution by electron transfer dissociation mass spectrometry. Anal. Chem. 81, 5577–5584 (2009).

    Article  CAS  PubMed  Google Scholar 

  76. Iacob, R. E. & Engen, J. R. Hydrogen exchange mass spectrometry: are we out of the quicksand? J. Am. Soc. Mass Spectrom. 23, 1003–1010 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Houde, D., Arndt, J., Domeier, W., Berkowitz, S. & Engen, J. R. Characterization of IgG1 conformation and conformational dynamics by hydrogen/deuterium exchange mass spectrometry. Anal. Chem. 81, 2644–2651 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Houde, D., Peng, Y., Berkowitz, S. A. & Engen, J. R. Post-translational modifications differentially affect IgG1 conformation and receptor binding. Mol. Cell Proteom. 9, 1716–1728 (2010).

    Article  CAS  Google Scholar 

  79. Kaltashov, I. A., Bobst, C. E., Abzalimov, R. R., Berkowitz, S. A. & Houde, D. Conformation and dynamics of biopharmaceuticals: transition of mass spectrometry-based tools from academe to industry. J. Am. Soc. Mass Spectrom. 21, 323–337 (2010).

    Article  CAS  PubMed  Google Scholar 

  80. Houde, D., Berkowitz, S. A. & Engen, J. R. The utility of hydrogen/deuterium exchange mass spectrometry in biopharmaceutical comparability studies. J. Pharm. Sci. 100, 2071–2086 (2011).

    Article  CAS  PubMed  Google Scholar 

  81. Wei, H. et al. Using hydrogen/deuterium exchange mass spectrometry to study conformational changes in granulocyte colony stimulating factor upon PEGylation. J. Am. Soc. Mass Spectrom. 23, 498–504 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Houde, D. & Demarest, S. J. Fine details of IGF-1R activation, inhibition, and asymmetry determined by associated hydrogen/deuterium-exchange and peptide mass mapping. Structure 19, 890–900 (2011).

    Article  CAS  PubMed  Google Scholar 

  83. Gerhardt, S. et al. Structure of IL-17A in complex with a potent, fully human neutralizing antibody. J. Mol. Biol. 394, 905–921 (2009).

    Article  CAS  PubMed  Google Scholar 

  84. Chalmers, M. J., Busby, S. A., Pascal, B. D., West, G. M. & Griffin, P. R. Differential hydrogen/deuterium exchange mass spectrometry analysis of protein–ligand interactions. Expert Rev. Proteom. 8, 43–59 (2011).

    Article  CAS  Google Scholar 

  85. Bagal, D., Valliere-Douglass, J. F., Balland, A. & Schnier, P. D. Resolving disulfide structural isoforms of IgG2 monoclonal antibodies by ion mobility mass spectrometry. Anal. Chem. 82, 6751–6755 (2010).

    Article  CAS  PubMed  Google Scholar 

  86. Wang, Y., Lu, Q., Wu, S. L., Karger, B. L. & Hancock, W. S. Characterization and comparison of disulfide linkages and scrambling patterns in therapeutic monoclonal antibodies: using LC-MS with electron transfer dissociation. Anal. Chem. 83, 3133–3140 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Wu, S. L., Jiang, H., Hancock, W. S. & Karger, B. L. Identification of the unpaired cysteine status and complete mapping of the 17 disulfides of recombinant tissue plasminogen activator using LC-MS with electron transfer dissociation/collision induced dissociation. Anal. Chem. 82, 5296–5303 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Benesch, J. L., Ruotolo, B. T., Simmons, D. A. & Robinson, C. V. Protein complexes in the gas phase: technology for structural genomics and proteomics. Chem. Rev. 107, 3544–3567 (2007).

    Article  CAS  PubMed  Google Scholar 

  89. Scarff, C. A., Thalassinos, K., Hilton, G. R. & Scrivens, J. H. Travelling wave ion mobility mass spectrometry studies of protein structure: biological significance and comparison with X-ray crystallography and nuclear magnetic resonance spectroscopy measurements. Rapid Commun. Mass Spectrom. 22, 3297–3304 (2008).

    Article  CAS  PubMed  Google Scholar 

  90. Zamani, L., Lindholm, J., Ilag, L. L. & Jacobsson, S. P. Discrimination among IgG1-κ monoclonal antibodies produced by two cell lines using charge state distributions in nanoESI-TOF mass spectra. J. Am. Soc. Mass Spectrom. 20, 1030–1036 (2009).

    Article  CAS  PubMed  Google Scholar 

  91. Bobst, C. E. & Kaltashov, I. A. Advanced mass spectrometry-based methods for the analysis of conformational integrity of biopharmaceutical products. Curr. Pharm. Biotechnol. 12, 1517–1529 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Bagal, D., Zhang, H. & Schnier, P. D. Gas-phase proton-transfer chemistry coupled with TOF mass spectrometry and ion mobility-MS for the facile analysis of poly(ethylene glycols) and PEGylated polypeptide conjugates. Anal. Chem. 80, 2408–2418 (2008).

    Article  CAS  PubMed  Google Scholar 

  93. Atmanene, C. et al. Extending mass spectrometry contribution to therapeutic monoclonal antibody lead optimization: characterization of immune complexes using noncovalent ESI-MS. Anal. Chem. 81, 6364–6373 (2009).

    Article  CAS  PubMed  Google Scholar 

  94. Kukrer, B. et al. Mass spectrometric analysis of intact human monoclonal antibody aggregates fractionated by size-exclusion chromatography. Pharm. Res. 27, 2197–2204 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Rand, K. D. et al. Gas-phase hydrogen/deuterium exchange in a traveling wave ion guide for the examination of protein conformations. Anal. Chem. 81, 10019–10028 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. Bucciantini, M. et al. Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature 416, 507–511 (2002).

    Article  CAS  PubMed  Google Scholar 

  97. Rosenberg, A. S. Effects of protein aggregates: an immunologic perspective. AAPS J. 8, E501–E507 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  98. Filipe, V., Hawe, A., Schellekens, H. & Jiskoot, W. in Aggregation of Therapeutic Proteins (eds Wang, W. & Roberts, C.J.) 400–433 (John Wiley and Sons, 2010).

    Google Scholar 

  99. Carpenter, J. F. et al. Overlooking subvisible particles in therapeutic protein products: gaps that may compromise product quality. J. Pharm. Sci. 98, 1201–1205 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Philo, J. S. A critical review of methods for size characterization of non-particulate protein aggregates. Curr. Pharm. Biotechnol. 10, 359–372 (2009).

    Article  CAS  PubMed  Google Scholar 

  101. Wang, W. & Roberts, C. J. (eds) Aggregation of Therapeutic Proteins (John Wiley and Sons, 2010). Reference 100 covers the main issues in protein aggregation and most of the key methodologies that have been developed to assess aggregation, whereas reference 101 provides a collection of articles that touch on all areas of therapeutic protein aggregation that are of great concern to the biopharmaceutical industry.

    Book  Google Scholar 

  102. Philo, J. S. Is any measurement method optimal for all aggregate sizes and types? AAPS J. 8, E564–E571 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  103. Mahler, H. C., Friess, W., Grauschopf, U. & Kiese, S. Protein aggregation: pathways, induction factors and analysis. J. Pharm. Sci. 98, 2909–2934 (2009).

    Article  CAS  PubMed  Google Scholar 

  104. Weiss, W. F., Young, T. M. & Roberts, C. J. Principles, approaches, and challenges for predicting protein aggregation rates and shelf life. J. Pharm. Sci. 98, 1246–1277 (2009).

    Article  CAS  PubMed  Google Scholar 

  105. Gabrielson, J. P., Arthur, K. K., Kendrick, B. S., Randolph, T. W. & Stoner, M. R. Common excipients impair detection of protein aggregates during sedimentation velocity analytical ultracentrifugation. J. Pharm. Sci. 98, 50–62 (2009).

    Article  CAS  PubMed  Google Scholar 

  106. Sharma, V. K. & Kalonia, D. S. in Aggregates of Therapeutic Proteins (eds Wang, W. & Roberts, C. J.) 205–256 (John Wiley and Sons, 2010).

    Book  Google Scholar 

  107. Cordoba-Rodriquez, R. V. Aggregates in MAbs and recombinant therapeutic proteins: a regulatory perspective. BioPharm. Int. 21, 44–53 (2008).

    Google Scholar 

  108. Arakawa, T., Philo, J. S., Ejima, D., Tsumoto, K. & Arisaka, F. Aggregation analysis of therapeutic proteins, part I: general aspects and techniques for assessment. BioProcess International 4, 32–42 (2006).

    CAS  Google Scholar 

  109. Arakawa, T., Philo, J. S., Ejima, D., Tsumoto, K. & Arisaka, F. Aggregation analysis of therapeutic proteins, part II: analytical ultracentrifugation and dynamic light scattering. BioProcess International 5, 36–47 (2007).

    CAS  Google Scholar 

  110. Arakawa, T., Philo, J. S., Ejima, D., Tsumoto, K. & Arisaka, F. Aggregation analysis of therapeutic proteins, part III: principles and optimization of field-flow fractionation (FFF). BioProcess International 5, 52–70 (2007).

    CAS  Google Scholar 

  111. Cromwell, M. E. M., Felten, C., Flores, H., Lui, J. & Shire, S. J. in Misbehaving Proteins: Protein (Mis)Folding, Aggregation, And Stability (eds Murphy, R. M. & Tsai, A. M.) 316–318 (Springer, 2006).

    Google Scholar 

  112. Demeule, B., Messick, S., Shire, S. J. & Liu, J. Characterization of particles in protein solutions: reaching the limits of current technologies. AAPS J. 12, 708–715 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  113. den Engelsman, J. et al. Strategies for the assessment of protein aggregates in pharmaceutical biotech product development. Pharm. Res. 28, 920–933 (2011).

    Article  CAS  PubMed  Google Scholar 

  114. Gabrielson, J. P. & Arthur, K. K. Measuring low levels of protein aggregation by sedimentation velocity. Methods 54, 83–91 (2011).

    Article  CAS  PubMed  Google Scholar 

  115. Berkowitz, S. A. Role of analytical ultracentrifugation in assessing the aggregation of protein biopharmaceuticals. AAPS J. 8, E590–E605 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  116. Jeong, J., Zhang, T., Zhang, J. & Kao, Y.-H. Ultra-high pressure LC (UHPLC) for therapeutic protein characterization. Amer. Pharma. Rev. 14, 44–51 (2011).

    CAS  Google Scholar 

  117. Arakawa, T., Ejima, D., Li, T. & Philo, J. S. The critical role of mobile phase composition in size exclusion chromatography of protein pharmaceuticals. J. Pharm. Sci. 99, 1674–1692 (2010).

    Article  CAS  PubMed  Google Scholar 

  118. Carpenter, J. F. et al. Potential inaccurate quantitation and sizing of protein aggregates by size exclusion chromatography: essential need to use orthogonal methods to assure the quality of therapeutic protein products. J. Pharm. Sci. 99, 2200–2208 (2010).

    Article  CAS  PubMed  Google Scholar 

  119. Gabrielson, J. P. et al. Quantitation of aggregate levels in a recombinant humanized monoclonal antibody formulation by size-exclusion chromatography, asymmetrical flow field flow fractionation, and sedimentation velocity. J. Pharm. Sci. 96, 268–279 (2007).

    Article  CAS  PubMed  Google Scholar 

  120. Liu, J., Andya, J. D. & Shire, S. J. A critical review of analytical ultracentrifugation and field flow fractionation methods for measuring protein aggregation. AAPS J. 8, E580–E589 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  121. Arthur, K. K., Gabrielson, J. P., Kendrick, B. S. & Stoner, M. R. Detection of protein aggregates by sedimentation velocity analytical ultracentrifugation (SV-AUC): sources of variability and their relative importance. J. Pharm. Sci. 98, 3522–3539 (2009).

    Article  CAS  PubMed  Google Scholar 

  122. Gabrielson, J. P. et al. Precision of protein aggregation measurements by sedimentation velocity analytical ultracentrifugation in biopharmaceutical applications. Anal. Biochem. 396, 231–241 (2010).

    Article  CAS  PubMed  Google Scholar 

  123. Gabrielson, J. P., Randolph, T. W., Kendrick, B. S. & Stoner, M. R. Sedimentation velocity analytical ultracentrifugation and SEDFIT/c(s): limits of quantitation for a monoclonal antibody system. Anal. Biochem. 361, 24–30 (2007).

    Article  CAS  PubMed  Google Scholar 

  124. Pekar, A. & Sukumar, M. Quantitation of aggregates in therapeutic proteins using sedimentation velocity analytical ultracentrifugation: practical considerations that affect precision and accuracy. Anal. Biochem. 367, 225–237 (2007).

    Article  CAS  PubMed  Google Scholar 

  125. Singh, S. K. et al. An industry perspective on the monitoring of subvisible particles as a quality attribute for protein therapeutics. J. Pharm. Sci. 99, 3302–3321 (2010).

    Article  CAS  PubMed  Google Scholar 

  126. Narhi, L. O., Jiang, Y., Cao, S., Benedek, K. & Shnek, D. A critical review of analytical methods for subvisible and visible particles. Curr. Pharm. Biotechnol. 10, 373–381 (2009).

    Article  CAS  PubMed  Google Scholar 

  127. Carpenter, J. et al. Meeting report on protein particles and immunogenicity of therapeutic proteins: filling in the gaps in risk evaluation and mitigation. Biologicals 38, 602–611 (2010).

    Article  PubMed  Google Scholar 

  128. Cao, X., Wen, Z. Q., Vance, A. & Torraca, G. Raman microscopic applications in the biopharmaceutical industry: in situ identification of foreign particulates inside glass containers with aqueous formulated solutions. Appl. Spectrosc. 63, 830–834 (2009).

    Article  CAS  PubMed  Google Scholar 

  129. Li, G., Torraca, G., Jing, W. & Wen, Z.-Q. Application of FTIR in identification of foreign materials for biopharmaceutical clinical manufacturing. Vibrat. Spectrosc. 50, 152–159 (2009).

    Article  CAS  Google Scholar 

  130. Rosenberg, A. & Worobec, A. A risk-based approach to immunogenicity concerns of therapeutic protein products, part I: considering consequences of the immune response to a protein. BioPharm Int. 17, 22–26 (2004).

    Google Scholar 

  131. Schellekens, H. Bioequivalence and the immunogenicity of biopharmaceuticals. Nature Rev. Drug Discov. 1, 457–462 (2002).

    Article  CAS  Google Scholar 

  132. Rosenberg, A. & Worobec, A. A risk-based approach to immunogenicity concerns of therapeutic protein products, part III: effects of manufacturing changes in immunogenicity and the utility of animal immunogenicity studies. BioPharm Int. 18, 32–36 (2005).

    Google Scholar 

  133. Sharma, B. Immunogenicity of therapeutic proteins. Part 1: impact of product handling. Biotechnol. Adv. 25, 310–317 (2007).

    Article  CAS  PubMed  Google Scholar 

  134. Sharma, B. Immunogenicity of therapeutic proteins. Part 2: impact of container closures. Biotechnol. Adv. 25, 318–324 (2007).

    Article  CAS  PubMed  Google Scholar 

  135. Sharma, B. Immunogenicity of therapeutic proteins. Part 3: impact of manufacturing changes. Biotechnol. Adv. 25, 325–331 (2007).

    Article  CAS  PubMed  Google Scholar 

  136. Richard, J. & Prang, N. The formulation and immunogenicity of therapeutic proteins: product quality as a key factor. IDrugs 13, 550–558 (2010).

    CAS  PubMed  Google Scholar 

  137. Rosenberg, A. & Worobec, A. A risk-based approach to immunogenicity concerns of therapeutic protein products, part II: considering host-specific and product-specific factors impacting immunogenicity. BioPharm Int. 17, 34–42 (2004).

    Google Scholar 

  138. Colfen, H. et al. The Open AUC Project. Eur. Biophys. J. 39, 347–359 (2010).

    Article  PubMed  Google Scholar 

  139. Zhang, Z. et al. Analysis of pharmaceutical heparins and potential contaminants using 1H-NMR and PAGE. J. Pharm. Sci. 98, 4017–4026 (2009).

    Article  CAS  PubMed  Google Scholar 

  140. United States Government Accountability Office (GAO). Food and Drug Administration: response to heparin contamination helped protect public health; controls that were needed for working with external entities were recently added. GAO website [online], (2010).

  141. European Medicines Agency. Biologics Working Party report: beta-interferons and neutralising antibodies (in multiple sclerosis). EMA website [online], (2008).

  142. Bernard Shaw, G. Man and Superman Act I (Brentano's, 1903).

    Google Scholar 

  143. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). Comparability of biotechnological/biological products subject to changes in their manufacturing process (ICH Q5E guidelines). ICH website [online], (2004).

  144. Chen, W., Chakraborty, A., Skilton, S. J., Berger, S. & Mazzeo, J. Characterizing biotherapeutic protein 3D structures by electrospray ion-mobility mass spectrometry: biological significance and comparison with X-ray crystallography and NMR measurements. Proceedings of the 58th ASMS Conference on Mass Spectrometry MOD 4:10 (23–27 May 2010; Salt Lake City, Utah, USA).

    Google Scholar 

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Acknowledgements

J.R.E. acknowledges research funding from the US National Institutes of Health (NIH) (R01-GM086507) and a research collaboration with the Waters Corporation. G.B.J. acknowledges research funding from the NIH (R01 CA111985-04), US Department of Energy (DE-SC0001781) and the US National Science Foundation (HRM 0811170). This is contribution 979 from the Barnett Institute.

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Authors and Affiliations

  1. Analytical Development, Biogen Idec, 14 Cambridge Center, Cambridge, 02142, Massachusetts, USA

    Steven A. Berkowitz

  2. Department of Chemistry & Chemical Biology, The Barnett Institute of Chemical & Biological Analysis, Northeastern University, 360 Huntington Ave., Boston, 02115, Massachusetts, USA

    John R. Engen & Graham B. Jones

  3. Waters Corporation, 34 Maple Street, Milford, 01757, Massachusetts, USA

    Jeffrey R. Mazzeo

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  1. Steven A. Berkowitz
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Correspondence to Graham B. Jones.

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Competing interests

Jeffrey R. Mazzeo is an employee of Waters Corporation, which manufactures chromatography and mass spectrometry instrumentation that can be used for protein analysis.

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FURTHER INFORMATION

Draft guidances for biosimilars on FDA website

EMA website (scientific guidance documents on biosimilar medicines)

FDA website — 27 October 2010 press release

rap.ID Particle Systems website

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Berkowitz, S., Engen, J., Mazzeo, J. et al. Analytical tools for characterizing biopharmaceuticals and the implications for biosimilars. Nat Rev Drug Discov 11, 527–540 (2012). https://doi.org/10.1038/nrd3746

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