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
Manning MC, Chou DK, Murphy BM, Payne RW, Katayama DS (2010) Stability of protein pharmaceuticals: an update. Pharm Res 27(4):544–575
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
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
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
Carpenter JF, Crowe JH (1989) An infrared spectroscopic study of the interactions of carbohydrates with dried proteins. Biochemistry 28(9):3916–3922
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
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
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
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
Iyer LK, Moorthy BS, Topp EM (2013) Photolytic labeling to probe molecular interactions in lyophilized powders. Mol Pharm 10(12):4629–4639
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
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
Wales TE, Engen JR (2006) Hydrogen exchange mass spectrometry for the analysis of protein dynamics. Mass Spectrom Rev 25(1):158–170
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
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
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
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
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
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
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
Moorthy BS, Iyer LK, Topp EM (2015) Characterizing protein structure, dynamics and conformation in lyophilized solids. Curr Pharm Des 21(40):5845–5853
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
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
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
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
Moorthy BS, Iyer LK, Topp EM (2015) Mass spectrometric approaches to study protein structure and interactions in lyophilized powders. J Vis Exp (98):52503
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
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
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
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
Brown KA, Wilson DJ (2017) Bottom-up hydrogen deuterium exchange mass spectrometry: data analysis and interpretation. Analyst 142(16):2874–2886
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
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
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
Hubbard RE, Kamran HM (2001) Hydrogen bonds in proteins: role and strength. In: eLS. Wiley, Chichester
Steiner T (2002) The hydrogen bond in the solid state. Angew Chem Int Ed 41(1):48–76
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
Crommelin DJA, Sindelar RD, Meibohm B (2013) Pharmaceutical biotechnology: fundamentals and applications. Springer Science & Business Media, New York
Pearl FMG, Sillitoe I, Orengo CA (2001) Protein structure classification. In: eLS. Wiley, Chichester
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
Fitzgerald MC, West GM (2009) Painting proteins with covalent labels: what’s in the picture? J Am Soc Mass Spectrom 20(6):1193–1206
Dubinsky L, Krom BP, Meijler MM (2012) Diazirine based photoaffinity labeling. Bioorg Med Chem 20(2):554–570
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
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
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
Jumper CC, Schriemer DC (2011) Mass spectrometry of laser-initiated carbene reactions for protein topographic analysis. Anal Chem 83(8):2913–2920
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
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
Hansen PR, Oddo A (2015) Fmoc solid-phase peptide synthesis. In: Houen G (ed) Peptide antibodies. Springer, New York, pp 33–50
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
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
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
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
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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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