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Lyophilization of Pharmaceuticals and Biologicals pp 353–375Cite as

High-Resolution Mass Spectrometric Methods for Proteins in Lyophilized Solids

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Part of the book series: Methods in Pharmacology and Toxicology ((MIPT))

Abstract

Lyophilization (freeze-drying) is used to produce amorphous solid powders of protein drugs. Though lyophilization is usually used in an attempt to stabilize the protein, degradation processes can still occur in the solid state, and are often poorly correlated with measurable properties of the protein and the powder. This chapter describes two novel, high-resolution mass-spectrometry-based methods for assessing protein structure and interactions in solid powders: solid-state hydrogen-deuterium exchange (ssHDX) and solid-state photolytic labeling (ssPLL) with mass spectrometric analysis (ssHDX-MS, ssPLL-MS). ssHDX-MS measures the rate of deuterium incorporation in the protein on exposure of the solid powder to D2O vapor. ssHDX-MS is thought to provide information regarding the network of inter- and intramolecular hydrogen bonds experienced by the protein in the solid state, and recent studies have shown that ssHDX metrics are highly correlated with stability on storage. ssPLL-MS provides complementary information on the protein’s side chain environment. The chapter summarizes the methods and recent results both ssHDX-MS and ssPLL-MS, and suggests directions for future research.

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References

  1. Manning MC, Chou DK, Murphy BM, Payne RW, Katayama DS (2010) Stability of protein pharmaceuticals: an update. Pharm Res 27(4):544–575

    Article  PubMed  CAS  Google Scholar 

  2. Lowe D, Dudgeon K, Rouet R, Schofield P, Jermutus L, Christ D (2011) Aggregation, stability, and formulation of human antibody therapeutics. Adv Protein Chem Struct Biol 84:41–61

    Article  CAS  PubMed  Google Scholar 

  3. Forney-Stevens KM, Bogner RH, Pikal MJ (2016) Addition of amino acids to further stabilize lyophilized sucrose-based protein formulations: I. Screening of 15 amino acids in two model proteins. J Pharm Sci 105(2):697–704

    Article  CAS  PubMed  Google Scholar 

  4. Ohtake S, Kita Y, Arakawa T (2011) Interactions of formulation excipients with proteins in solution and in the dried state. Adv Drug Deliv Rev 63(13):1053–1073

    Article  CAS  PubMed  Google Scholar 

  5. Carpenter JF, Crowe JH (1989) An infrared spectroscopic study of the interactions of carbohydrates with dried proteins. Biochemistry 28(9):3916–3922

    Article  CAS  PubMed  Google Scholar 

  6. Duddu SP, Zhang G, Dal Monte PR (1997) The relationship between protein aggregation and molecular mobility below the glass transition temperature of lyophilized formulations containing a monoclonal antibody. Pharm Res 14(5):596–600

    Article  CAS  PubMed  Google Scholar 

  7. Zhang Z, Smith DL (1993) Determination of amide hydrogen exchange by mass spectrometry: a new tool for protein structure elucidation. Protein Sci 2(4):522–531

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Moorthy BS, Zarraga IE, Kumar L, Walters BT, Goldbach P, Topp EM et al (2018) Solid-state hydrogen–deuterium exchange mass spectrometry: correlation of deuterium uptake and long-term stability of lyophilized monoclonal antibody formulations. Mol Pharm 15(1):1–11

    Article  CAS  PubMed  Google Scholar 

  9. Moorthy BS, Schultz SG, Kim SG, Topp EM (2014) Predicting protein aggregation during storage in lyophilized solids using solid state amide hydrogen/deuterium exchange with mass spectrometric analysis (ssHDX-MS). Mol Pharm 11(6):1869–1879

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Iyer LK, Moorthy BS, Topp EM (2013) Photolytic labeling to probe molecular interactions in lyophilized powders. Mol Pharm 10(12):4629–4639

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Iyer LK, Moorthy BS, Topp EM (2015) Photolytic cross-linking to probe protein–protein and protein–matrix interactions in lyophilized powders. Mol Pharm 12(9):3237–3249

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Iyer LK, Sacha GA, Moorthy BS, Nail SL, Topp EM (2016) Process and formulation effects on protein structure in lyophilized solids using mass spectrometric methods. J Pharm Sci 105(5):1684–1692

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Wales TE, Engen JR (2006) Hydrogen exchange mass spectrometry for the analysis of protein dynamics. Mass Spectrom Rev 25(1):158–170

    Article  CAS  PubMed  Google Scholar 

  14. Tsutsui Y, Wintrode PL (2007) Hydrogen/deuterium exchange-mass spectrometry: a powerful tool for probing protein structure, dynamics and interactions. Curr Med Chem 14(22):2344–2358

    Article  CAS  PubMed  Google Scholar 

  15. Houde D, Berkowitz SA, Engen JR (2011) The utility of hydrogen/deuterium exchange mass spectrometry in biopharmaceutical comparability studies. J Pharm Sci 100(6):2071–2086

    Article  CAS  PubMed  Google Scholar 

  16. Majumdar R, Middaugh CR, Weis DD, Volkin DB (2015) Hydrogen-deuterium exchange mass spectrometry as an emerging analytical tool for stabilization and formulation development of therapeutic monoclonal antibodies. J Pharm Sci 104(2):327–345

    Article  CAS  PubMed  Google Scholar 

  17. Li Y, Williams TD, Schowen RL, Topp EM (2007) Trehalose and calcium exert site-specific effects on calmodulin conformation in amorphous solids. Biotechnol Bioeng 97(6):1650–1653

    Article  CAS  PubMed  Google Scholar 

  18. Sinha S, Li Y, Williams TD, Topp EM (2008) Protein conformation in amorphous solids by FTIR and by hydrogen/deuterium exchange with mass spectrometry. Biophys J 95(12):5951–5961

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Sophocleous AM, Topp EM (2012) Localized hydration in lyophilized myoglobin by hydrogen-deuterium exchange mass spectrometry. 2. Exchange kinetics. Mol Pharm 9(4):727–733

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Sophocleous AM, Zhang J, Topp EM (2012) Localized hydration in lyophilized myoglobin by hydrogen–deuterium exchange mass spectrometry. 1. Exchange mapping. Mol Pharm 9(4):718–726

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Moorthy BS, Iyer LK, Topp EM (2015) Characterizing protein structure, dynamics and conformation in lyophilized solids. Curr Pharm Des 21(40):5845–5853

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Moussa EM, Singh SK, Kimmel M, Nema S, Topp EM (2018) Probing the conformation of an IgG1 monoclonal antibody in lyophilized solids using solid-state hydrogen–deuterium exchange with mass spectrometric analysis (ssHDX-MS). Mol Pharm 15(2):356–368

    Article  CAS  PubMed  Google Scholar 

  23. Moussa EM, Wilson NE, Zhou QT, Singh SK, Nema S, Topp EM (2018) Effects of drying process on an IgG1 monoclonal antibody using solid-state hydrogen deuterium exchange with mass spectrometric analysis (ssHDX-MS). Pharm Res 35(1):12

    Article  PubMed  CAS  Google Scholar 

  24. Li Y, Williams TD, Schowen RL, Topp EM (2007) Characterizing protein structure in amorphous solids using hydrogen/deuterium exchange with mass spectrometry. Anal Biochem 366(1):18–28

    Article  CAS  PubMed  Google Scholar 

  25. Li Y, Williams TD, Topp EM (2008) Effects of excipients on protein conformation in lyophilized solids by hydrogen/deuterium exchange mass spectrometry. Pharm Res 25(2):259–267

    Article  CAS  PubMed  Google Scholar 

  26. Moorthy BS, Iyer LK, Topp EM (2015) Mass spectrometric approaches to study protein structure and interactions in lyophilized powders. J Vis Exp (98):52503

    Google Scholar 

  27. Sane SU, Wong R, Hsu CC (2004) Raman spectroscopic characterization of drying-induced structural changes in a therapeutic antibody: correlating structural changes with long-term stability. J Pharm Sci 93(4):1005–1018

    Article  CAS  PubMed  Google Scholar 

  28. Chang LL, Shepherd D, Sun J, Ouellette D, Grant KL, Tang XC et al (2005) Mechanism of protein stabilization by sugars during freeze-drying and storage: native structure preservation, specific interaction, and/or immobilization in a glassy matrix? J Pharm Sci 94(7):1427–1444

    Article  CAS  PubMed  Google Scholar 

  29. Schüle S, Frieß W, Bechtold-Peters K, Garidel P (2007) Conformational analysis of protein secondary structure during spray-drying of antibody/mannitol formulations. Eur J Pharm Biopharm 65(1):1–9

    Article  PubMed  CAS  Google Scholar 

  30. Park J, Nagapudi K, Vergara C, Ramachander R, Laurence JS, Krishnan S (2013) Effect of pH and excipients on structure, dynamics, and long-term stability of a model IgG1 monoclonal antibody upon freeze-drying. Pharm Res 30(4):968–984

    Article  CAS  PubMed  Google Scholar 

  31. Brown KA, Wilson DJ (2017) Bottom-up hydrogen deuterium exchange mass spectrometry: data analysis and interpretation. Analyst 142(16):2874–2886

    Article  CAS  PubMed  Google Scholar 

  32. Konermann L, Pan J, Liu Y-H (2011) Hydrogen exchange mass spectrometry for studying protein structure and dynamics. Chem Soc Rev 40(3):1224–1234

    Article  CAS  PubMed  Google Scholar 

  33. Deng B, Lento C, Wilson DJ (2016) Hydrogen deuterium exchange mass spectrometry in biopharmaceutical discovery and development–a review. Anal Chim Acta 940:8–20

    Article  CAS  PubMed  Google Scholar 

  34. Huang RY-C, Chen G (2014) Higher order structure characterization of protein therapeutics by hydrogen/deuterium exchange mass spectrometry. Anal Bioanal Chem 406(26):6541–6558

    Article  CAS  PubMed  Google Scholar 

  35. Hubbard RE, Kamran HM (2001) Hydrogen bonds in proteins: role and strength. In: eLS. Wiley, Chichester

    Google Scholar 

  36. Steiner T (2002) The hydrogen bond in the solid state. Angew Chem Int Ed 41(1):48–76

    Article  CAS  Google Scholar 

  37. Koehl P (2006) Protein structure classification. In: Lipkowitz KB, Cundari TR, Gillet VJ, Boyd DB (eds) Reviews in computational chemistry. Wiley, Chichester, pp 1–55

    Google Scholar 

  38. Crommelin DJA, Sindelar RD, Meibohm B (2013) Pharmaceutical biotechnology: fundamentals and applications. Springer Science & Business Media, New York

    Book  Google Scholar 

  39. Pearl FMG, Sillitoe I, Orengo CA (2001) Protein structure classification. In: eLS. Wiley, Chichester

    Google Scholar 

  40. Breen ED, Curley JG, Overcashier DE, Hsu CC, Shire SJ (2001) Effect of moisture on the stability of a lyophilized humanized monoclonal antibody formulation. Pharm Res 18(9):1345–1353

    Article  CAS  PubMed  Google Scholar 

  41. Fitzgerald MC, West GM (2009) Painting proteins with covalent labels: what’s in the picture? J Am Soc Mass Spectrom 20(6):1193–1206

    Article  CAS  PubMed  Google Scholar 

  42. Dubinsky L, Krom BP, Meijler MM (2012) Diazirine based photoaffinity labeling. Bioorg Med Chem 20(2):554–570

    Article  CAS  PubMed  Google Scholar 

  43. Chin JW, Martin AB, King DS, Wang L, Schultz PG (2002) Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli. Proc Natl Acad Sci U S A 99(17):11020–11024

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Hino N, Okazaki Y, Kobayashi T, Hayashi A, Sakamoto K, Yokoyama S (2005) Protein photo-cross-linking in mammalian cells by site-specific incorporation of a photoreactive amino acid. Nat Methods 2(3):201

    Article  CAS  PubMed  Google Scholar 

  45. Suchanek M, Radzikowska A, Thiele C (2005) Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells. Nat Methods 2(4):261

    Article  CAS  PubMed  Google Scholar 

  46. Jumper CC, Schriemer DC (2011) Mass spectrometry of laser-initiated carbene reactions for protein topographic analysis. Anal Chem 83(8):2913–2920

    Article  CAS  PubMed  Google Scholar 

  47. Kölbel K, Ihling CH, Sinz A (2012) Analysis of peptide secondary structures by photoactivatable amino acid analogues. Angew Chem Int Ed 51(50):12602–12605

    Article  CAS  Google Scholar 

  48. Brodie NI, Makepeace KAT, Petrotchenko EV, Borchers CH (2015) Isotopically-coded short-range hetero-bifunctional photo-reactive crosslinkers for studying protein structure. J Proteome 118:12–20

    Article  CAS  Google Scholar 

  49. Hansen PR, Oddo A (2015) Fmoc solid-phase peptide synthesis. In: Houen G (ed) Peptide antibodies. Springer, New York, pp 33–50

    Chapter  Google Scholar 

  50. Farrell IS, Toroney R, Hazen JL, Mehl RA, Chin JW (2005) Photo-cross-linking interacting proteins with a genetically encoded benzophenone. Nat Methods 2(5):377

    Article  CAS  PubMed  Google Scholar 

  51. Venditti V, Fawzi NL, Clore GM (2012) An efficient protocol for incorporation of an unnatural amino acid in perdeuterated recombinant proteins using glucose-based media. J Biomol NMR 52(3):191–195

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Young TS, Ahmad I, Yin JA, Schultz PG (2010) An enhanced system for unnatural amino acid mutagenesis in E. coli. J Mol Biol 395(2):361–374

    Article  CAS  PubMed  Google Scholar 

  53. Janz JM, Ren Y, Looby R, Kazmi MA, Sachdev P, Grunbeck A et al (2011) Direct interaction between an allosteric agonist pepducin and the chemokine receptor CXCR4. J Am Chem Soc 133(40):15878–15881

    Article  CAS  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Industrial and Physical Pharmacy, Purdue University, West Lafayette, IN, USA

    Karthik Balakrishna Chandrababu, Rajashekar Kammari, Yuan Chen & Elizabeth M. Topp

Authors
  1. Karthik Balakrishna Chandrababu
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  2. Rajashekar Kammari
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  3. Yuan Chen
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  4. Elizabeth M. Topp
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Corresponding author

Correspondence to Elizabeth M. Topp .

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Editors and Affiliations

  1. Biopharma Process Systems Ltd., Winchester, UK

    Kevin R. Ward

  2. National Institute for Biological Standards & Control (NIBSC), Potters Bar, Hertfordshire, UK

    Paul Matejtschuk

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Balakrishna Chandrababu, K., Kammari, R., Chen, Y., Topp, E.M. (2019). High-Resolution Mass Spectrometric Methods for Proteins in Lyophilized Solids. In: Ward, K., Matejtschuk, P. (eds) Lyophilization of Pharmaceuticals and Biologicals. Methods in Pharmacology and Toxicology. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8928-7_14

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  • DOI: https://doi.org/10.1007/978-1-4939-8928-7_14

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8927-0

  • Online ISBN: 978-1-4939-8928-7

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