The AAPS Journal

, Volume 16, Issue 1, pp 48–64 | Cite as

High-Throughput Biophysical Analysis of Protein Therapeutics to Examine Interrelationships Between Aggregate Formation and Conformational Stability

  • Rajoshi Chaudhuri
  • Yuan Cheng
  • C. Russell Middaugh
  • David B. Volkin
Mini-Review Theme: Aggregation and Interactions of Therapeutic Proteins


Stabilization and formulation of therapeutic proteins against physical instability, both structural alterations and aggregation, is particularly challenging not only due to each protein’s unique physicochemical characteristics but also their susceptibility to the surrounding milieu (pH, ionic strength, excipients, etc.) as well as various environmental stresses (temperature, agitation, lyophilization, etc.). The use of high-throughput techniques can significantly aid in the evaluation of stabilizing solution conditions by permitting a more rapid evaluation of a large matrix of possible combinations. In this mini-review, we discuss both key physical degradation pathways observed for protein-based drugs and the utility of various high-throughput biophysical techniques to aid in protein formulation development to minimize their occurrence. We then focus on four illustrative case studies with therapeutic protein candidates of varying sizes, shapes and physicochemical properties to explore different analytical challenges in monitoring protein physical instability. These include an IgG2 monoclonal antibody, an albumin-fusion protein, a recombinant pentameric plasma glycoprotein, and an antibody fragment (Fab). Future challenges and opportunities to improve and apply high-throughput approaches to protein formulation development are also discussed.


aggregation biophysical conformation formulation mini-review monoclonal antibody protein stability structure 


  1. 1.
    Mullard A. 2010 FDA drug approvals. Nat Rev Drug Discov. 2011;10:82–5.PubMedGoogle Scholar
  2. 2.
    Reichert JM. Marketed therapeutic antibodies compendium. mAbs. 2012;4:413–5.PubMedCentralPubMedGoogle Scholar
  3. 3.
    Reissner KJ, Aswad DW. Deamidation and isoaspartate formation in proteins: unwanted alterations or surreptitious signals? Cell Mol Life Sci. 2003;60:1281–95.PubMedGoogle Scholar
  4. 4.
    Lam XM, Yang JY, Cleland JL. Antioxidants for prevention of methionine oxidation in recombinant monoclonal antibody HER2. J Pharm Sci. 1997;86:1250–5.PubMedGoogle Scholar
  5. 5.
    Manning MC, Chou DK, Murphy BM, Payne RW, Katayama DS. Stability of protein pharmaceuticals: an update. Pharm Res. 2010;27:544–75.PubMedGoogle Scholar
  6. 6.
    Wang W, Singh SK, Li N, Toler MR, King KR, Nema S. Immunogenicity of protein aggregates–concerns and realities. Int J Pharm. 2012;431:1–11.PubMedGoogle Scholar
  7. 7.
    Fandrich M, Schmidt M, Grigorieff N. Recent progress in understanding Alzheimer’s beta-amyloid structures. Trends Biochem Sci. 2011;36:338–45.PubMedCentralPubMedGoogle Scholar
  8. 8.
    Tovey MG, Legrand J, Lallemand C. Overcoming immunogenicity associated with the use of biopharmaceuticals. Expert Rev Clin Pharmacol. 2011;4:623–31.PubMedGoogle Scholar
  9. 9.
    Wang W, Nema S, Teagarden D. Protein aggregation–pathways and influencing factors. Int J Pharm. 2010;390:89–99.PubMedGoogle Scholar
  10. 10.
    Voynov V, Chennamsetty N, Kayser V, Helk B, Trout BL. Predictive tools for stabilization of therapeutic proteins. mAbs. 2009;1:580–2.PubMedCentralPubMedGoogle Scholar
  11. 11.
    Wang W, Roberts CJ. Non-Arrhenius protein aggregation. The AAPS J. 2013;15:840–51.Google Scholar
  12. 12.
    Narhi LO, Schmit J, Bechtold-Peters K, Sharma D. Classification of protein aggregates. J Pharm Sci. 2012;101:493–8.PubMedGoogle Scholar
  13. 13.
    Bond MD, Panek ME, Zhang Z, Wang D, Mehndiratta P, Zhao H, et al. Evaluation of a dual-wavelength size exclusion HPLC method with improved sensitivity to detect protein aggregates and its use to better characterize degradation pathways of an IgG1 monoclonal antibody. J Pharm Sci. 2010;99:2582–97.PubMedGoogle Scholar
  14. 14.
    Wuchner K, Buchler J, Spycher R, Dalmonte P, Volkin DB. Development of a microflow digital imaging assay to characterize protein particulates during storage of a high concentration IgG1 monoclonal antibody formulation. J Pharm Sci. 2010;99:3343–61.PubMedGoogle Scholar
  15. 15.
    Roberts CJ, Das TK, Sahin E. Predicting solution aggregation rates for therapeutic proteins: approaches and challenges. Int J Pharm. 2011;418:318–33.PubMedGoogle Scholar
  16. 16.
    Volkin DB, Mach H, Middaugh CR. Degradative covalent reactions important to protein stability. Mol Biotechnol. 1997;8:105–22.PubMedGoogle Scholar
  17. 17.
    Liu D, Ren D, Huang H, Dankberg J, Rosenfeld R, Cocco MJ, et al. Structure and stability changes of human IgG1 Fc as a consequence of methionine oxidation. Biochemistry. 2008;47:5088–100.PubMedGoogle Scholar
  18. 18.
    Tsai AM, van Zanten JH, Betenbaugh MJ. I. Study of protein aggregation due to heat denaturation: a structural approach using circular dichroism spectroscopy, nuclear magnetic resonance, and static light scattering. Biotech Bioeng. 1998;59:273–80.Google Scholar
  19. 19.
    Cheng W, Joshi SB, He F, Brems DN, He B, Kerwin BA, et al. Comparison of high-throughput biophysical methods to identify stabilizing excipients for a model IgG2 monoclonal antibody: conformational stability and kinetic aggregation measurements. J Pharm Sci. 2012;101:1701–20.PubMedGoogle Scholar
  20. 20.
    Andersen CB, Manno M, Rischel C, Thorolfsson M, Martorana V. Aggregation of a multidomain protein: a coagulation mechanism governs aggregation of a model IgG1 antibody under weak thermal stress. Protein Sci Publ Prote Soc. 2010;19:279–90.Google Scholar
  21. 21.
    Perico N, Purtell J, Dillon TM, Ricci MS. Conformational implications of an inversed pH-dependent antibody aggregation. J Pharm Sci. 2009;98:3031–42.PubMedGoogle Scholar
  22. 22.
    Vermeer AW, Norde W. The thermal stability of immunoglobulin: unfolding and aggregation of a multi-domain protein. Biophys J. 2000;78:394–404.PubMedCentralPubMedGoogle Scholar
  23. 23.
    Ejima D, Tsumoto K, Fukada H, Yumioka R, Nagase K, Arakawa T, et al. Effects of acid exposure on the conformation, stability, and aggregation of monoclonal antibodies. Proteins. 2007;66:954–62.PubMedGoogle Scholar
  24. 24.
    Kroetsch AM, Sahin E, Wang HY, Krizman S, Roberts CJ. Relating particle formation to salt- and pH-dependent phase separation of non-native aggregates of alpha-chymotrypsinogen A. J Pharm Sci. 2012;101:3651–60.PubMedGoogle Scholar
  25. 25.
    Sahin E, Weiss WFT, Kroetsch AM, King KR, Kessler RK, Das TK, et al. Aggregation and pH-temperature phase behavior for aggregates of an IgG2 antibody. J Pharm Sci. 2012;101:1678–87.PubMedGoogle Scholar
  26. 26.
    Wang W, Singh S, Zeng DL, King K, Nema S. Antibody structure, instability, and formulation. J Pharm Sci. 2007;96:1–26.PubMedGoogle Scholar
  27. 27.
    Hamada H, Arakawa T, Shiraki K. Effect of additives on protein aggregation. Curr Pharm Biotechnol. 2009;10:400–7.PubMedGoogle Scholar
  28. 28.
    Kendrick BS, Carpenter JF, Cleland JL, Randolph TW. A transient expansion of the native state precedes aggregation of recombinant human interferon-gamma. Proc Natl Acad Sci U S A. 1998;95:14142–6.PubMedCentralPubMedGoogle Scholar
  29. 29.
    Deyoung LR, Fink AL, Dill KA. Aggregation of globular-proteins. Acc Chem Res. 1993;26:614–20.Google Scholar
  30. 30.
    Guo B, Kao S, McDonald H, Asanov A, Combs LL, Wilson WW. Correlation of second virial coefficients and solubilities useful in protein crystal growth. J Cryst Growth. 1999;196:424–33.Google Scholar
  31. 31.
    Valente JJ, Payne RW, Manning MC, Wilson WW, Henry CS. Colloidal behavior of proteins: effects of the second virial coefficient on solubility, crystallization and aggregation of proteins in aqueous solution. Curr Pharm Biotechnol. 2005;6:427–36.PubMedGoogle Scholar
  32. 32.
    Saluja A, Kalonia DS. Nature and consequences of protein–protein interactions in high protein concentration solutions. Int J Pharm. 2008;358:1–15.PubMedGoogle Scholar
  33. 33.
    Szenczi A, Kardos J, Medgyesi GA, Zavodszky P. The effect of solvent environment on the conformation and stability of human polyclonal IgG in solution. Biologicals J Int Assoc Biol Stand. 2006;34:5–14.Google Scholar
  34. 34.
    Olsen SN, Andersen KB, Randolph TW, Carpenter JF, Westh P. Role of electrostatic repulsion on colloidal stability of Bacillus halmapalus alpha-amylase. Biochim Biophys Acta. 2009;1794:1058–65.PubMedGoogle Scholar
  35. 35.
    Sahin E, Grillo AO, Perkins MD, Roberts CJ. Comparative effects of pH and ionic strength on protein–protein interactions, unfolding, and aggregation for IgG1 antibodies. J Pharm Sci. 2010;99:4830–48.PubMedGoogle Scholar
  36. 36.
    Rubin J, Linden L, Coco WM, Bommarius AS, Behrens SH. Salt-induced aggregation of a monoclonal human immunoglobulin G1. J Pharm Sci. 2013;102:377–86.PubMedGoogle Scholar
  37. 37.
    Zhang-van Enk J, Mason BD, Yu L, Zhang L, Hamouda W, Huang G, et al. Perturbation of thermal unfolding and aggregation of human IgG1 Fc fragment by Hofmeister anions. Mol Pharm. 2013;10:619–30.PubMedGoogle Scholar
  38. 38.
    Fesinmeyer RM, Hogan S, Saluja A, Brych SR, Kras E, Narhi LO, et al. Effect of ions on agitation- and temperature-induced aggregation reactions of antibodies. Pharm Res. 2009;26:903–13.PubMedGoogle Scholar
  39. 39.
    Gokarn YR, Fesinmeyer RM, Saluja A, Razinkov V, Chase SF, Laue TM, et al. Effective charge measurements reveal selective and preferential accumulation of anions, but not cations, at the protein surface in dilute salt solutions. Protein Sci Publ Protein Soc. 2011;20:580–7.Google Scholar
  40. 40.
    Majumdar R, Manikwar P, Hickey JM, Samra HS, Sathish HA, Bishop SM, et al. Effects of salts from the Hofmeister series on the conformational stability, aggregation propensity, and local flexibility of an IgG1 monoclonal antibody. Biochemistry. 2013;52:3376–89.PubMedGoogle Scholar
  41. 41.
    Manikwar P, Majumdar R, Hickey JM, Thakkar SV, Samra HS, Sathish HA, et al. Correlating excipient effects on conformational and storage stability of an IgG1 monoclonal antibody with local dynamics as measured by hydrogen/deuterium-exchange mass spectrometry. J Pharm Sci. 2013;102:2136–51.PubMedGoogle Scholar
  42. 42.
    Krishnan S, Chi EY, Wood SJ, Kendrick BS, Li C, Garzon-Rodriguez W, et al. Oxidative dimer formation is the critical rate-limiting step for Parkinson’s disease alpha-synuclein fibrillogenesis. Biochemistry. 2003;42:829–37.PubMedGoogle Scholar
  43. 43.
    Come JH, Fraser PE, Lansbury Jr PT. A kinetic model for amyloid formation in the prion diseases: importance of seeding. Proc Natl Acad Sci U S A. 1993;90:5959–63.PubMedCentralPubMedGoogle Scholar
  44. 44.
    Navarra G, Troia F, Militello V, Leone M. Characterization of the nucleation process of lysozyme at physiological pH: primary but not sole process. Biophys Chem. 2013;177–178:24–33.PubMedGoogle Scholar
  45. 45.
    Chi EY, Weickmann J, Carpenter JF, Manning MC, Randolph TW. Heterogeneous nucleation-controlled particulate formation of recombinant human platelet-activating factor acetylhydrolase in pharmaceutical formulation. J Pharm Sci. 2005;94:256–74.PubMedGoogle Scholar
  46. 46.
    Bajaj H, Sharma VK, Badkar A, Zeng D, Nema S, Kalonia DS. Protein structural conformation and not second virial coefficient relates to long-term irreversible aggregation of a monoclonal antibody and ovalbumin in solution. Pharm Res. 2006;23:1382–94.PubMedGoogle Scholar
  47. 47.
    Chi EY, Krishnan S, Kendrick BS, Chang BS, Carpenter JF, Randolph TW. Roles of conformational stability and colloidal stability in the aggregation of recombinant human granulocyte colony-stimulating factor. Protein Sci Publ Protein Soc. 2003;12:903–13.Google Scholar
  48. 48.
    Lowe D, Dudgeon K, Rouet R, Schofield P, Jermutus L, Christ D. Aggregation, stability, and formulation of human antibody therapeutics. Adv Protein Chem Struct Biol. 2011;84:41–61.PubMedGoogle Scholar
  49. 49.
    Kim N, Remmele Jr RL, Liu D, Razinkov VI, Fernandez EJ, Roberts CJ. Aggregation of anti-streptavidin immunoglobulin gamma-1 involves Fab unfolding and competing growth pathways mediated by pH and salt concentration. Biophys Chem. 2013;172:26–36.PubMedGoogle Scholar
  50. 50.
    Saito S, Hasegawa J, Kobayashi N, Tomitsuka T, Uchiyama S, Fukui K. Effects of ionic strength and sugars on the aggregation propensity of monoclonal antibodies: influence of colloidal and conformational stabilities. Pharm Res. 2013;30:1263–80.PubMedGoogle Scholar
  51. 51.
    Arosio P, Rima S, Morbidelli M. Aggregation mechanism of an IgG2 and two IgG1 monoclonal antibodies at low pH: from oligomers to larger aggregates. Pharm Res. 2013;30:641–54.PubMedGoogle Scholar
  52. 52.
    Goldberg DS, Bishop SM, Shah AU, Sathish HA. Formulation development of therapeutic monoclonal antibodies using high-throughput fluorescence and static light scattering techniques: role of conformational and colloidal stability. J Pharm Sci. 2011;100:1306–15.PubMedGoogle Scholar
  53. 53.
    Gibson TJ, McCarty K, McFadyen IJ, Cash E, Dalmonte P, Hinds KD, et al. Application of a high-throughput screening procedure with PEG-induced precipitation to compare relative protein solubility during formulation development with IgG1 monoclonal antibodies. J Pharm Sci. 2011;100:1009–21.PubMedGoogle Scholar
  54. 54.
    He F, Phan DH, Hogan S, Bailey R, Becker GW, Narhi LO, et al. Detection of IgG aggregation by a high throughput method based on extrinsic fluorescence. J Pharm Sci. 2010;99:2598–608.PubMedGoogle Scholar
  55. 55.
    He F, Woods CE, Becker GW, Narhi LO, Razinkov VI. High-throughput assessment of thermal and colloidal stability parameters for monoclonal antibody formulations. J Pharm Sci. 2011;100:5126–41.PubMedGoogle Scholar
  56. 56.
    Samra HS, He F. Advancements in high throughput biophysical technologies: applications for characterization and screening during early formulation development of monoclonal antibodies. Mol Pharm. 2012;9:696–707.PubMedGoogle Scholar
  57. 57.
    Kamerzell TJ, Esfandiary R, Joshi SB, Middaugh CR, Volkin DB. Protein-excipient interactions: mechanisms and biophysical characterization applied to protein formulation development. Adv Drug Deliv Rev. 2011;63:1118–59.PubMedGoogle Scholar
  58. 58.
    Razinkov VI, Treuheit MJ, Becker GW. Methods of high throughput biophysical characterization in biopharmaceutical development. Curr Drug Discov Technol. 2013;10:59–70.PubMedGoogle Scholar
  59. 59.
    Freire E. Differential scanning calorimetry. Methods Mol Biol. 1995;40:191–218.PubMedGoogle Scholar
  60. 60.
    Bedu-Addo FK, Johnson C, Jeyarajah S, Henderson I, Advant SJ. Use of biophysical characterization in preformulation development of a heavy-chain fragment of botulinum serotype B: evaluation of suitable purification process conditions. Pharm Res. 2004;21:1353–61.PubMedGoogle Scholar
  61. 61.
    Ishikawa T, Ito T, Endo R, Nakagawa K, Sawa E, Wakamatsu K. Influence of pH on heat-induced aggregation and degradation of therapeutic monoclonal antibodies. Biol Pharm Bull. 2010;33:1413–7.PubMedGoogle Scholar
  62. 62.
    Bhugra C, Pikal MJ. Role of thermodynamic, molecular, and kinetic factors in crystallization from the amorphous state. J Pharm Sci. 2008;97:1329–49.PubMedGoogle Scholar
  63. 63.
    Wang L, Wang B, Lin Q. Demonstration of MEMS-based differential scanning calorimetry for determing thermodynamic properties of biomolecules. Sens Actuators B-Chem. 2008; 134(2):953–958.Google Scholar
  64. 64.
    Malik K, Matejtschuk P, Thelwell C, Burns CJ. Differential scanning fluorimetry: rapid screening of formulations that promote the stability of reference preparations. J Pharm Biomed Anal. 2013;77:163–6.PubMedGoogle Scholar
  65. 65.
    Menzen T, Friess W. High-throughput melting-temperature analysis of a monoclonal antibody by differential scanning fluorimetry in the presence of surfactants. J Pharm Sci. 2013;102:415–28.PubMedGoogle Scholar
  66. 66.
    He F, Hogan S, Latypov RF, Narhi LO, Razinkov VI. High throughput thermostability screening of monoclonal antibody formulations. J Pharm Sci. 2010;99:1707–20.PubMedGoogle Scholar
  67. 67.
    Hawe A, Filipe V, Jiskoot W. Fluorescent molecular rotors as dyes to characterize polysorbate-containing IgG formulations. Pharm Res. 2010;27:314–26.PubMedCentralPubMedGoogle Scholar
  68. 68.
    Capelle MA, Gurny R, Arvinte T. A high throughput protein formulation platform: case study of salmon calcitonin. Pharm Res. 2009;26:118–28.PubMedGoogle Scholar
  69. 69.
    Garidel P, Hegyi M, Bassarab S, Weichel M. A rapid, sensitive and economical assessment of monoclonal antibody conformational stability by intrinsic tryptophan fluorescence spectroscopy. Biotechnol J. 2008;3:1201–11.PubMedGoogle Scholar
  70. 70.
    Maddux NR, Joshi SB, Volkin DB, Ralston JP, Middaugh CR. Multidimensional methods for the formulation of biopharmaceuticals and vaccines. J Pharm Sci. 2011;100:4171–97.Google Scholar
  71. 71.
    Maddux NR, Rosen IT, Hu L, Olsen CM, Volkin DB, Middaugh CR. An improved methodology for multidimensional high-throughput preformulation characterization of protein conformational stability. J Pharm Sci. 2012;101:2017–24.PubMedGoogle Scholar
  72. 72.
    Hawe A, Sutter M, Jiskoot W. Extrinsic fluorescent dyes as tools for protein characterization. Pharm Res. 2008;25:1487–99.PubMedCentralPubMedGoogle Scholar
  73. 73.
    Hu L, Joshi SB, Liyanage MR, Pansalawatta M, Alderson MR, Tate A, et al. Physical characterization and formulation development of a recombinant pneumolysoid protein-based pneumococcal vaccine. J Pharm Sci. 2013;102:387–400.PubMedGoogle Scholar
  74. 74.
    Iyer V, Maddux N, Hu L, Cheng W, Youssef AK, Winter G, et al. Comparative signature diagrams to evaluate biophysical data for differences in protein structure across various formulations. J Pharm Sci. 2013;102:43–51.PubMedGoogle Scholar
  75. 75.
    Hu L, Olsen C, Maddux N, Joshi SB, Volkin DB, Middaugh CR. Investigation of protein conformational stability employing a multimodal spectrometer. Analytical chemistry. 2011;83:9399–405.PubMedGoogle Scholar
  76. 76.
    Schmitz K. An introduction to dynamic light scattering by macromolecules. New York: Academic; 1990.Google Scholar
  77. 77.
    Ahrer K, Buchacher A, Iberer G, Jungbauer A. Thermodynamic stability and formation of aggregates of human immunoglobulin G characterised by differential scanning calorimetry and dynamic light scattering. J Biochem Biophys Methods. 2006;66:73–86.PubMedGoogle Scholar
  78. 78.
    Nobbmann U, Connah M, Fish B, Varley P, Gee C, Mulot S, et al. Dynamic light scattering as a relative tool for assessing the molecular integrity and stability of monoclonal antibodies. Biotechnol Genet Eng Rev. 2007;24:117–28.PubMedGoogle Scholar
  79. 79.
    Ahrer K, Buchacher A, Iberer G, Josic D, Jungbauer A. Analysis of aggregates of human immunoglobulin G using size-exclusion chromatography, static and dynamic light scattering. J Chromatogr A. 2003;1009:89–96.PubMedGoogle Scholar
  80. 80.
    He F, Becker GW, Litowski JR, Narhi LO, Brems DN, Razinkov VI. High-throughput dynamic light scattering method for measuring viscosity of concentrated protein solutions. Anal Biochem. 2010;399:141–3.PubMedGoogle Scholar
  81. 81.
    Attri AK, Minton AP. New methods for measuring macromolecular interactions in solution via static light scattering: basic methodology and application to nonassociating and self-associating proteins. Anal Biochem. 2005;337:103–10.PubMedGoogle Scholar
  82. 82.
    Esfandiary R, Hayes DB, Parupudi A, Casas-Finet J, Bai S, Samra HS, et al. A systematic multitechnique approach for detection and characterization of reversible self-association during formulation development of therapeutic antibodies. J Pharm Sci. 2013;102:62–72.PubMedGoogle Scholar
  83. 83.
    Sahin E, Roberts CJ. Size-exclusion chromatography with multi-angle light scattering for elucidating protein aggregation mechanisms. Methods Mol Biol. 2012;899:403–23.PubMedGoogle Scholar
  84. 84.
    D’Souza AJ, Ford BM, Mar KD, Sullivan VJ. Biophysical characterization and formulation of F1-V, a recombinant plague antigen. J Pharm Sci. 2009;98:2592–602.PubMedGoogle Scholar
  85. 85.
    Volkin DB, Tsai PK, Dabora JM, Gress JO, Burke CJ, Linhardt RJ, et al. Physical stabilization of acidic fibroblast growth factor by polyanions. Arch Biochem Biophys. 1993;300:30–41.PubMedGoogle Scholar
  86. 86.
    Salinas BA, Sathish HA, Bishop SM, Harn N, Carpenter JF, Randolph TW. Understanding and modulating opalescence and viscosity in a monoclonal antibody formulation. J Pharm Sci. 2010;99:82–93.PubMedGoogle Scholar
  87. 87.
    Tantipolphan R, Romeijn S, Engelsman J, Torosantucci R, Rasmussen T, Jiskoot W. Elution behavior of insulin on high-performance size exclusion chromatography at neutral pH. J Pharm Biomed Anal. 2010;52:195–202.PubMedGoogle Scholar
  88. 88.
    Philo JS. Is any measurement method optimal for all aggregate sizes and types? AAPS J. 2006;8:E564–71.PubMedCentralPubMedGoogle Scholar
  89. 89.
    Bajaj H, Sharma VK, Kalonia DS. A high-throughput method for detection of protein self-association and second virial coefficient using size-exclusion chromatography through simultaneous measurement of concentration and scattered light intensity. Pharm Res. 2007;24:2071–83.PubMedGoogle Scholar
  90. 90.
    Berkowitz SA. Role of analytical ultracentrifugation in assessing the aggregation of protein biopharmaceuticals. The AAPS J. 2006;8:E590–605.Google Scholar
  91. 91.
    Shi S, Liu J, Joshi SB, Krasnoperov V, Gill P, Middaugh CR, et al. Biophysical characterization and stabilization of the recombinant albumin fusion protein sEphB4-HSA. J Pharm Sci. 2012;101:1969–84.PubMedGoogle Scholar
  92. 92.
    Scehnet JS, Ley EJ, Krasnoperov V, Liu R, Manchanda PK, Sjoberg E, et al. The role of Ephs, Ephrins, and growth factors in Kaposi sarcoma and implications of EphrinB2 blockade. Blood. 2009;113:254–63.PubMedGoogle Scholar
  93. 93.
    Liu J, Blasie CA, Shi S, Joshi SB, Middaugh CR, Volkin DB. Characterization and stabilization of recombinant human protein pentraxin (rhPTX-2). J Pharm Sci. 2013;102:827–41.PubMedGoogle Scholar
  94. 94.
    Bottazzi B, Garlanda C, Salvatori G, Jeannin P, Manfredi A, Mantovani A. Pentraxins as a key component of innate immunity. Curr Opin Immunol. 2006;18:10–5.PubMedGoogle Scholar
  95. 95.
    Emsley J, White HE, O’Hara BP, Oliva G, Srinivasan N, Tickle IJ, et al. Structure of pentameric human serum amyloid P component. Nature. 1994;367:338–45.PubMedGoogle Scholar
  96. 96.
    Duffield JS, Lupher Jr ML. PRM-151 (recombinant human serum amyloid P/pentraxin 2) for the treatment of fibrosis. Drug News Perspect. 2010;23:305–15.PubMedGoogle Scholar
  97. 97.
    Wang T, Kumru OS, Yi L, Wang YJ, Zhang J, Kim JH. Effect of ionic strength and pH on the physical and chemical stability of a monoclonal antibody antigen-binding fragment. Can J Pharm Sci. 2013; 102(8):2520-37Google Scholar
  98. 98.
    Zolls S, Tantipolphan R, Wiggenhorn M, Winter G, Jiskoot W, Friess W, et al. Particles in therapeutic protein formulations, part 1: overview of analytical methods. J Pharm Sci. 2012;101:914–35.PubMedGoogle Scholar
  99. 99.
    Kim JH, Iyer V, Joshi SB, Volkin DB, Middaugh CR. Improved data visualization techniques for analyzing macromolecule structural changes. Protein Sci Publ Prote Soc. 2012;21:1540–53.Google Scholar
  100. 100.
    Federici M, Lubiniecki A, Manikwar P, Volkin DB. Analytical lessons learned from selected therapeutic protein drug comparability studies. Biologicals J Int Assoc Biol Stand. 2013;41:131–47.Google Scholar

Copyright information

© American Association of Pharmaceutical Scientists 2013

Authors and Affiliations

  • Rajoshi Chaudhuri
    • 1
    • 2
  • Yuan Cheng
    • 1
  • C. Russell Middaugh
    • 1
  • David B. Volkin
    • 1
  1. 1.Department of Pharmaceutical Chemistry, Macromolecule and Vaccine Stabilization CenterUniversity of KansasLawrenceUSA
  2. 2.Vaccine Production Program Laboratory, Vaccine Research Center/NIAIDNational Institutes of Health, DHHSGaithersburgUSA

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