Chapter 7: An Empirical Phase Diagram: High-Throughput Screening Approach to the Characterization and Formulation of Biopharmaceuticals

  • Yangjie Wei
  • Sangeeta B. Joshi
  • Akhilesh Bhambhani
  • Yuhong Zeng
  • Nicholas R. Larson
  • Gang Hu
  • Eric J. Deeds
  • C. Russell MiddaughEmail author
Part of the AAPS Advances in the Pharmaceutical Sciences Series book series (AAPS, volume 35)


The complexity of proteins usually requires the use of multiple, moderate resolution experimental techniques to provide an information-rich picture of their structure and stability. Here we describe a technique known as the empirical phase diagram (EPD), which can be used to present such multidimensional data in picture form. Data from various spectroscopic, hydrodynamic, and calorimetric methods, among others, are created in matrix form and converted to a colored picture as a function of variables such as temperature, pH, ionic strength, etc. Additional methods such as radar charts and Chernoff faces are also discussed. The use of EPDs in the characterization and formulation of biomolecules is described. Further methods based on machine learning are also considered. A wide range of examples are employed to illustrate the power and utility of these approaches.


Empirical phase diagram (EPD) Biologics Formulation screening Stability Biophysical characterization Radar chart Chernoff face Machine learning 


  1. 1.
    Joshi SB, Bhambhani A, Zeng Y, Middaugh CR. An empirical phase diagram–high-throughput screening approach to the characterization and formulation of biopharmaceuticals. In: Jameel F, Hershenson S, editors. Formulation and process development strategies for manufacturing biopharmaceuticals. Hoboken: Wiley; 2010. p. 173–205.CrossRefGoogle Scholar
  2. 2.
    Chang BS, Hershenson S. Practical approaches to protein formulation development. In: Carpenter JF, Manning MC, editors. Rational design of stable protein formulations: theory and practice. Parmaceutical viotechnology, vol. 13. New York: Kluwer Academic; 2002. p. 1–25.CrossRefGoogle Scholar
  3. 3.
    Venyaminov SY, Yang JT. Determination of protein secondary structure. In: Fasman GD, editor. Circular dichroism and the conformational analysis of biomolecules. New York: Plenum Press; 1996. p. 69–107.CrossRefGoogle Scholar
  4. 4.
    Fan H, Vitharana SN, Chen T, O’Keefe D, Middaugh CR. Effects of pH and polyanions on the thermal stability of fibroblast growth factor 20. Mol Pharm. 2007;4(2):232–40.PubMedCrossRefPubMedCentralGoogle Scholar
  5. 5.
    Ausar SA, Joshi SB, Middaugh CR. Spectroscopic methods for the physical characterization and formulation of nonviral gene delivery systems. In: Le Doux J, editor. Gene therapy protocols. Methods in molecular biology. 3rd ed. Totowa: Humana Press, Inc.; 2008. p. 55–80.CrossRefGoogle Scholar
  6. 6.
    Braun CS, Kueltzo LA, Middaugh CR. Ultraviolet absorption and circular dichroism spectroscopy of nonviral gene delivery complexes. In: Findeis MA, editor. Nonviral vectors for gene therapy: methods and protocols. Totowa: Humana Press Inc.; 2001. p. 253–84.CrossRefGoogle Scholar
  7. 7.
    Jiskoot W, Visser AJWG, Herron JN, Sutter M. Fluorescence spectroscopy. In: Jiskoot W, Crommelin DJA, editors. Methods for structural analysis of protein pharmaceuticals. Biotechnology: pharmaceutical aspects. III: American association of pharmaceutical scientists. New York: AAPS Press; 2005. p. 27–82.Google Scholar
  8. 8.
    Stryer L. The interaction of a naphthalene dye with apomyoglobin and apohemoglobin. A fluorescent probe of non-polar binding sites. J Mol Biol. 1965;13(2):482–95.PubMedCrossRefPubMedCentralGoogle Scholar
  9. 9.
    Cardamone M, Puri NK. Spectrofluorimetric assessment of the surface hydrophobicity of proteins. Biochem J. 1992;282(Pt 2):589–93.PubMedPubMedCentralCrossRefGoogle Scholar
  10. 10.
    Ali V, Prakash K, Kulkarni S, Ahmad A, Madhusudan KP, Bhakuni V. 8-Anilino-1-naphthalene sulfonic acid (ANS) induces folding of acid unfolded cytochrome c to molten globule state as a result of electrostatic interactions. Biochemistry. 1999;38(41):13635–42.PubMedCrossRefPubMedCentralGoogle Scholar
  11. 11.
    Fan H, Kashi RS, Middaugh CR. Conformational lability of two molecular chaperones Hsc70 and GP96: effects of pH and temperature. Arch Biochem Biophys. 2006;447(1):34–45.PubMedCrossRefPubMedCentralGoogle Scholar
  12. 12.
    Matulis D, Lovrien R. 1-Anilino-8-naphthalene sulfonate anion-protein binding depends primarily on ion pair formation. Biophys J. 1998;74(1):422–9.PubMedPubMedCentralCrossRefGoogle Scholar
  13. 13.
    Klunk WE, Pettegrew JW, Abraham DJ. Quantitative evaluation of congo red binding to amyloid-like proteins with a beta-pleated sheet conformation. J Histochem Cytochem. 1989;37(8):1273–81.PubMedCrossRefPubMedCentralGoogle Scholar
  14. 14.
    Guntern R, Bouras C, Hof PR, Vallet PG. An improved thioflavine S method for staining neurofibrillary tangles and senile plaques in Alzheimer’s disease. Experientia. 1992;48(1):8–10.PubMedCrossRefPubMedCentralGoogle Scholar
  15. 15.
    LeVine H 3rd. Thioflavine T interaction with synthetic Alzheimer’s disease beta-amyloid peptides: detection of amyloid aggregation in solution. Protein Sci. 1993;2(3):404–10.PubMedPubMedCentralCrossRefGoogle Scholar
  16. 16.
    LeVine H 3rd. Quantification of beta-sheet amyloid fibril structures with thioflavin T. Methods Enzymol. 1999;309:274–84.PubMedCrossRefPubMedCentralGoogle Scholar
  17. 17.
    Vassar PS, Culling CF. Fluorescent stains, with special reference to amyloid and connective tissues. Arch Pathol. 1959;68:487–98.PubMedPubMedCentralGoogle Scholar
  18. 18.
    Alonso LG, Garcia-Alai MM, Smal C, Centeno JM, Iacono R, Castano E, et al. The HPV16 E7 viral oncoprotein self-assembles into defined spherical oligomers. Biochemistry. 2004;43(12):3310–7.PubMedCrossRefPubMedCentralGoogle Scholar
  19. 19.
    Hatters DM, MacPhee CE, Lawrence LJ, Sawyer WH, Howlett GJ. Human apolipoprotein C-II forms twisted amyloid ribbons and closed loops. Biochemistry. 2000;39(28):8276–83.PubMedCrossRefPubMedCentralGoogle Scholar
  20. 20.
    Wiethoff CM, Middaugh CR. Light-scattering techniques for characterization of synthetic gene therapy vectors. In: Findeis MA, editor. Nonviral vectors for gene therapy: methods and protocols. Totowa: Humana Press Ind.; 2001. p. 349–76.CrossRefGoogle Scholar
  21. 21.
    Freire E. Differential scanning calorimetry. Methods Mol Biol. 1995;40:191–218.PubMedPubMedCentralGoogle Scholar
  22. 22.
    Kueltzo LA, Ersoy B, Darrington T, Ralston JP, Middaugh CR. Derivative absorbance spectroscopy and protein phase diagrams as tools for comprehensive protein characterization: a bGCSF case study. J Pharm Sci. 2003;92(9):1805–20.PubMedCrossRefPubMedCentralGoogle Scholar
  23. 23.
    Kueltzo LA, Middaugh CR. Structural characterization of bovine granulocyte colony stimulating factor: effect of temperature and pH. J Pharm Sci. 2003;92(9):1793–804.PubMedCrossRefPubMedCentralGoogle Scholar
  24. 24.
    Fan H, Ralston J, DiBase M, Faulkner E, Middaugh CR. Solution behavior of IFN-ß-1a: an empirical phase diagram based approach. J Pharm Sci. 2005;94(9):1893–911.PubMedCrossRefPubMedCentralGoogle Scholar
  25. 25.
    Jiang G, Joshi SB, Peek LJ, Brandau DT, Huang CR, Ferriter MS, et al. Anthrax vaccine powder formulations for nasal mucosal delivery. J Pharm Sci. 2006;95(1):80–96.PubMedCrossRefPubMedCentralGoogle Scholar
  26. 26.
    Peek LJ, Brey RN, Middaugh CR. A rapid, three-step process for the preformulation of a recombinant ricin toxin A-chain vaccine. J Pharm Sci. 2007;96(1):44–60.PubMedCrossRefPubMedCentralGoogle Scholar
  27. 27.
    Fan H, Li H, Zhang M, Middaugh CR. Effects of solutes on empirical phase diagrams of human fibroblast growth factor 1. J Pharm Sci. 2007;96(6):1490–503.PubMedCrossRefPubMedCentralGoogle Scholar
  28. 28.
    Brandau DT, Joshi SB, Smalter AM, Kim S, Steadman B, Middaugh CR. Stability of the Clostridium botulinum type A neurotoxin complex: an empirical phase diagram based approach. Mol Pharm. 2007;4(4):571–82.PubMedCrossRefPubMedCentralGoogle Scholar
  29. 29.
    Ausar SF, Rexroad J, Frolov VG, Look JL, Konar N, Middaugh CR. Analysis of the thermal and pH stability of human respiratory syncytial virus. Mol Pharm. 2005;2(6):491–9.PubMedCrossRefPubMedCentralGoogle Scholar
  30. 30.
    Ausar SF, Foubert TR, Hudson MH, Vedvick TS, Middaugh CR. Conformational stability and disassembly of Norwalk virus like particles: effect of pH and temperature. J Biol Chem. 2006;281(28):19478–88.PubMedCrossRefPubMedCentralGoogle Scholar
  31. 31.
    Harn N, Allan C, Oliver C, Middaugh CR. Highly concentrated monoclonal antibodies: direct analysis of structure and stability. J Pharm Sci. 2007;96(3):532–46.PubMedCrossRefPubMedCentralGoogle Scholar
  32. 32.
    Peek LJ, Brandau DT, Jones LS, Joshi SB, Middaugh CR. A systematic approach to stabilizing EBA-175 RII-NG for use as a malaria vaccine. Vaccine. 2006;24(31–32):5839–51.PubMedCrossRefPubMedCentralGoogle Scholar
  33. 33.
    Rexroad J, Martin TT, McNeilly D, Godwin S, Middaugh CR. Thermal stability of adenovirus type 2 as a function of pH. J Pharm Sci. 2006;95(7):1469–79.PubMedCrossRefPubMedCentralGoogle Scholar
  34. 34.
    Ruponen M, Braun CS, Middaugh CR. Biophysical characterization of polymeric and liposomal gene delivery systems using empirical phase diagrams. J Pharm Sci. 2006;95(10):2101–14.PubMedCrossRefPubMedCentralGoogle Scholar
  35. 35.
    Thyagrajapuram N, Olsen D, Middaugh CR. The structure stability and complex behavior of recombinant human gelatins. J Pharm Sci. 2007;96:3363–78.CrossRefGoogle Scholar
  36. 36.
    Nonoyama A, Laurence JS, Garriques L, Qi H, Le T, Middaugh CR. A biophysical characterization of the peptide drug pramlintide (AC137) using empirical phase diagrams. J Pharm Sci. 2008;97(7):2552–67.PubMedCrossRefPubMedCentralGoogle Scholar
  37. 37.
    Salnikova MS, Joshi SB, Rytting JH, Warny M, Middaugh CR. Physical characterization of Clostridium difficile toxins and toxoids: effect of the formaldehyde crosslinking on thermal stability. J Pharm Sci. 2008;97(9):3735–52.PubMedCrossRefPubMedCentralGoogle Scholar
  38. 38.
    Rexroad J, Evans RK, Middaugh CR. Effect of pH and ionic strength on the physical stability of adenovirus type 5. J Pharm Sci. 2006;95(2):237–47.PubMedCrossRefPubMedCentralGoogle Scholar
  39. 39.
    Shire SJ, Shahrokh Z, Liu J. Challenges in the development of high protein concentration formulations. J Pharm Sci. 2004;93(6):1390–402.PubMedCrossRefPubMedCentralGoogle Scholar
  40. 40.
    Zimmer C, Luck G, venner H. Studies on the conformation of protonated DNA. Biopolymers. 1968;6:563–4.PubMedCrossRefPubMedCentralGoogle Scholar
  41. 41.
    Kamerzell TJ, Middaugh CR. Two-dimensional correlation spectroscopy reveals coupled immunoglobulin regions of differential flexibility that influence stability. Biochemistry. 2007;46(34):9762–73.PubMedCrossRefPubMedCentralGoogle Scholar
  42. 42.
    Miller DW, Dill KA. A statistical mechanical model for hydrogen exchange in globular proteins. Protein Sci. 1995;4(9):1860–73.PubMedPubMedCentralCrossRefGoogle Scholar
  43. 43.
    Woodward CK, Hilton BD. Hydrogen exchange kinetics and internal motions in proteins and nucleic acids. Annu Rev Biophys Bioeng. 1979;8:99–127.PubMedCrossRefPubMedCentralGoogle Scholar
  44. 44.
    Majumdar R, Middaugh CR, Weis DD, Volkin DB. Hydrogen-deuterium exchange mass spectrometry as an emerging analytical tool for stabilization and formulation development of therapeutic monoclonal antibodies. J Pharm Sci. 2015;104(2):327–45.PubMedCrossRefPubMedCentralGoogle Scholar
  45. 45.
    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(7):2136–51.PubMedCrossRefPubMedCentralGoogle Scholar
  46. 46.
    Joshi SB, Kamerzell TJ, McNown C, Middaugh CR. The interaction of heparin/polyanions with bovine, porcine, and human growth hormone. J Pharm Sci. 2008;97(4):1368–85.PubMedCrossRefPubMedCentralGoogle Scholar
  47. 47.
    Eftink M, Ghiron C. Dynamics of a protein matrix revealed by fluorescence quenching. Proc Natl Acad Sci U S A. 1975;72:3290–4.PubMedPubMedCentralCrossRefGoogle Scholar
  48. 48.
    Kamerzell TJ, Unruh JR, Johnson CK, Middaugh CR. Conformational flexibility, hydration and state parameter fluctuations of fibroblast growth factor-10: effects of ligand binding. Biochemistry. 2006;45:15288–300.PubMedCrossRefPubMedCentralGoogle Scholar
  49. 49.
    Thakkar SV, Joshi SB, Jones ME, Sathish HA, Bishop SM, Volkin DB, et al. Excipients differentially influence the conformational stability and pretransition dynamics of two IgG1 monoclonal antibodies. J Pharm Sci. 2012;101(9):3062–77.PubMedCrossRefPubMedCentralGoogle Scholar
  50. 50.
    Ramsey JD, Gill ML, Kamerzell TJ, Price ES, Joshi SB, Bishop SM, et al. Using empirical phase diagrams to understand the role of intramolecular dynamics in immunoglobulin G stability. J Pharm Sci. 2009;98(7):2432–47.PubMedPubMedCentralCrossRefGoogle Scholar
  51. 51.
    Kim JH, Iyer V, Joshi SB, Volkin DB, Middaugh CR. Improved data visualization techniques for analyzing macromolecule structural changes. Protein Sci. 2012;21(10):1540–53.PubMedPubMedCentralCrossRefGoogle Scholar
  52. 52.
    Kim JH, Joshi SB, Tolbert TJ, Middaugh CR, Volkin DB, Hall AS. Biosimilarity assessments of model IgG1-Fc glycoforms using a machine learning approach. J Pharm Sci. 2016;105(2):602–12.PubMedCrossRefPubMedCentralGoogle Scholar
  53. 53.
    Nariya MK, Kim JH, Xiong J, Kleindl PA, Hewarathna A, Fisher AC, et al. Comparative characterization of crofelemer samples using data mining and machine learning approaches with analytical stability data sets. J Pharm Sci. 2017;106(11):3270–9.PubMedPubMedCentralCrossRefGoogle Scholar
  54. 54.
    Hewarathna A, Mozziconacci O, Nariya MK, Kleindl PA, Xiong J, Fisher AC, et al. Chemical stability of the botanical drug substance crofelemer: a model system for comparative characterization of complex mixture drugs. J Pharm Sci. 2017;106(11):3257–69.PubMedPubMedCentralCrossRefGoogle Scholar
  55. 55.
    Kleindl PA, Xiong J, Hewarathna A, Mozziconacci O, Nariya MK, Fisher AC, et al. The botanical drug substance crofelemer as a model system for comparative characterization of complex mixture drugs. J Pharm Sci. 2017;106(11):3242–56.PubMedPubMedCentralCrossRefGoogle Scholar
  56. 56.
    Okbazghi SZ, More AS, White DR, Duan S, Shah IS, Joshi SB, et al. Production, characterization, and biological evaluation of well-defined IgG1 Fc glycoforms as a model system for biosimilarity analysis. J Pharm Sci. 2016;105(2):559–74.PubMedPubMedCentralCrossRefGoogle Scholar
  57. 57.
    More AS, Toprani VM, Okbazghi SZ, Kim JH, Joshi SB, Middaugh CR, et al. Correlating the impact of well-defined oligosaccharide structures on physical stability profiles of IgG1-Fc glycoforms. J Pharm Sci. 2016;105(2):588–601.PubMedCrossRefPubMedCentralGoogle Scholar
  58. 58.
    Cover TM, Thomas JA. Elements of information theory. 2nd ed. Hoboken: Wiley-Interscience; 2006.Google Scholar
  59. 59.
    Castro JG, Chin-Beckford N. Crofelemer for the symptomatic relief of non-infectious diarrhea in adult patients with HIV/AIDS on anti-retroviral therapy. Expert Rev Clin Pharmacol. 2015;8(6):683–90.PubMedCrossRefPubMedCentralGoogle Scholar
  60. 60.
    Maclean DS, Qian Q, Middaugh CR. Stabilization of proteins by low molecular weight multi-ions. J Pharm Sci. 2002;91(10):2220–9.PubMedCrossRefPubMedCentralGoogle Scholar
  61. 61.
    Yoshioka S, Stella VJ. Stability of drugs and dosage forms. New York: Kluwer Academic/Plenum Publishers; 2000. p. 187–99.Google Scholar
  62. 62.
    Goldberg DS, Lewus RA, Esfandiary R, Farkas DC, Mody N, Day KJ, et al. Utility of high throughput screening techniques to predict stability of monoclonal antibody formulations during early stage development. J Pharm Sci. 2017;106(8):1971–7.PubMedCrossRefPubMedCentralGoogle Scholar
  63. 63.
    Ausar SF, Espina M, Brock J, Thyagarayapuran N, Repetto R, Khandke L, et al. High-through put screening of stabilizers for respiratory syncytial virus: identification of stabilizers and their effects on the conformational thermostability of viral particles. Hum Vaccin. 2007;3(3):94–103.PubMedCrossRefPubMedCentralGoogle Scholar
  64. 64.
    Salnikova MS, Joshi SB, Rytting JH, Warny M, Middaugh CR. Preformulation studies of Clostridium difficile toxoids A and B. J Pharm Sci. 2008;97(10):4197–207.CrossRefGoogle Scholar
  65. 65.
    He F, Joshi SB, Moore DD, Shinogle HE, Ohtake S, Lechuga-Ballesteros D, et al. Using spectroscopic and microscopic methods to probe the structural stability of human adenovirus type 4. Hum Vaccin. 2010;6(2):202–11.PubMedCrossRefPubMedCentralGoogle Scholar
  66. 66.
    Kissmann J, Ausar SF, Rudolph A, Braun C, Cape SP, Sievers RE, et al. Stabilization of measles virus for vaccine formulation. Hum Vaccin. 2008;4(5):350–9.PubMedCrossRefPubMedCentralGoogle Scholar
  67. 67.
    Esfandiary R, Yee L, Ohtake S, Martin RA, Truong-Le VL, Lechuga-Ballesteros D, et al. Biophysical characterization of rotavirus serotypes G1, G3, and G4. Hum Vaccin. 2010;6(5):390–8.PubMedCrossRefPubMedCentralGoogle Scholar
  68. 68.
    Esfandiary R, Kickhoefer VA, Rome LH, Joshi SB, Middaugh CR. Structural stability of vault particles. J Pharm Sci. 2009;98(4):1376–86.PubMedCrossRefPubMedCentralGoogle Scholar
  69. 69.
    Zeng Y, Fan H, Chiueh G, Pham B, Martin R, Lechuga-Ballesteros D, et al. Towards development of stable formulations of a live attenuated bacterial vaccine: a preformulation study facilitated by a biophysical approach. Hum Vaccin. 2009;5(5):322–31.PubMedCrossRefPubMedCentralGoogle Scholar
  70. 70.
    Harn N, Allan C, Oliver C, Middaugh C. Highly concentrated monoclonal antibody solutions: direct analysis of physical structure and thermal stability. J Pharm Sci. 2007;96(3):532–46.PubMedCrossRefPubMedCentralGoogle Scholar
  71. 71.
    Barrett BS, Picking WL, Picking WD, Middaugh CR. The response of type three secretion system needle proteins MxiHΔ5, BsaLΔ5, and PrgIΔ5 to temperature and pH. Proteins. 2008;73(3):632–43.PubMedCrossRefPubMedCentralGoogle Scholar
  72. 72.
    Markham AP, Jaafar ZA, Kemege KE, Middaugh CR, Hefty PS. Biophysical characterization of Chlamydia trachomatis CT584 supports its potential role as a type III secretion needle tip protein. Biochemistry. 2009;48(43):10353–61.PubMedPubMedCentralCrossRefGoogle Scholar
  73. 73.
    Cai S, He F, Samra HS, de la Maza LM, Bottazzi ME, Joshi SB, et al. Biophysical and stabilization studies of the Chlamydia trachomatis mouse pneumonitis major outer membrane protein. Mol Pharm. 2009;6(5):1553–61.PubMedPubMedCentralCrossRefGoogle Scholar
  74. 74.
    Zheng K, Middaugh CR, Siahaan TJ. Evaluation of the physical stability of the EC5 domain of E-cadherin: effects of pH, temperature, ionic strength, and disulfide bonds. J Pharm Sci. 2009;98(1):63–73.PubMedPubMedCentralCrossRefGoogle Scholar
  75. 75.
    Hu L, Olsen C, Maddux N, Joshi SB, Volkin DB, Middaugh CR. Investigation of protein conformational stability employing a multimodal spectrometer. Anal Chem. 2011;83(24):9399–405.PubMedCrossRefPubMedCentralGoogle Scholar
  76. 76.
    Hu L, Trefethen JM, Zeng Y, Yee L, Ohtake S, Lechuga-Ballesteros D, et al. Biophysical characterization and conformational stability of Ebola and Marburg virus-like particles. J Pharm Sci. 2011;100(12):5156–73.PubMedCrossRefPubMedCentralGoogle Scholar
  77. 77.
    Kissmann J, Joshi SB, Haynes JR, Dokken L, Richardson C, Middaugh CR. H1N1 influenza virus-like particles: physical degradation pathways and identification of stabilizers. J Pharm Sci. 2011;100(2):634–45.PubMedCrossRefPubMedCentralGoogle Scholar
  78. 78.
    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(6):1969–84.PubMedCrossRefPubMedCentralGoogle Scholar
  79. 79.
    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(6):2017–24.PubMedPubMedCentralCrossRefGoogle Scholar
  80. 80.
    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(5):1701–20.PubMedCrossRefPubMedCentralGoogle Scholar
  81. 81.
    Plieskatt J, Rezende W, Olsen C, Trefethen J, Joshi SB, Middaugh CR, et al. Advances in vaccines against neglected tropical diseases: enhancing physical stability of a recombinant hookworm vaccine through biophysical and formulation studies. Hum Vaccin Immunother. 2012;8(6):765–76.PubMedCrossRefPubMedCentralGoogle Scholar
  82. 82.
    Iyer V, Liyanage MR, Shoji Y, Chichester JA, Jones RM, Yusibov V, et al. Formulation development of a plant-derived h1n1 influenza vaccine containing purified recombinant hemagglutinin antigen. Hum Vaccin Immunother. 2012;8(4):453–64.PubMedCrossRefPubMedCentralGoogle Scholar
  83. 83.
    Hu L, Joshi SB, Andra KK, Thakkar SV, Volkin DB, Bann JG, et al. Comparison of the structural stability and dynamic properties of recombinant anthrax protective antigen and its 2-fluorohistidine-labeled analogue. J Pharm Sci. 2012;101(11):4118–28.PubMedPubMedCentralCrossRefGoogle Scholar
  84. 84.
    Iyer V, Hu L, Liyanage MR, Esfandiary R, Reinisch C, Meinke A, et al. Preformulation characterization of an aluminum salt-adjuvanted trivalent recombinant protein-based vaccine candidate against streptococcus pneumoniae. J Pharm Sci. 2012;101(9):3078–90.PubMedCrossRefPubMedCentralGoogle Scholar
  85. 85.
    Alsenaidy MA, Wang T, Kim JH, Joshi SB, Lee J, Blaber M, et al. An empirical phase diagram approach to investigate conformational stability of “second-generation” functional mutants of acidic fibroblast growth factor-1. Protein Sci. 2012;21(3):418–32.PubMedCrossRefPubMedCentralGoogle Scholar
  86. 86.
    Bhambhani A, Kissmann JM, Joshi SB, Volkin DB, Kashi RS, Middaugh CR. Formulation design and high-throughput excipient selection based on structural integrity and conformational stability of dilute and highly concentrated IgG1 monoclonal antibody solutions. J Pharm Sci. 2012;101(3):1120–35.PubMedCrossRefPubMedCentralGoogle Scholar
  87. 87.
    Alsenaidy MA, Kim JH, Majumdar R, Weis DD, Joshi SB, Tolbert TJ, et al. High-throughput biophysical analysis and data visualization of conformational stability of an IgG1 monoclonal antibody after deglycosylation. J Pharm Sci. 2013;102(11):3942–56.PubMedCrossRefPubMedCentralGoogle Scholar
  88. 88.
    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(3):827–41.PubMedCrossRefPubMedCentralGoogle Scholar
  89. 89.
    Wang T, Kumru OS, Yi L, Wang YJ, Zhang J, Kim JH, et al. Effect of ionic strength and pH on the physical and chemical stability of a monoclonal antibody antigen-binding fragment. J Pharm Sci. 2013;102(8):2520–37.PubMedCrossRefPubMedCentralGoogle Scholar
  90. 90.
    Hamborg M, Kramer R, Schanté CE, Agger EM, Christensen D, Jorgensen L, et al. The physical stability of the recombinant tuberculosis fusion antigens H 1 and H 56. J Pharm Sci. 2013;102(10):3567–78.PubMedCrossRefPubMedCentralGoogle Scholar
  91. 91.
    Choudhari SP, Kramer R, Barta ML, Greenwood JC, Geisbrecht BV, Joshi SB, et al. Studies of the conformational stability of invasion plasmid antigen B from Shigella. Protein Sci. 2013;22(5):666–70.PubMedPubMedCentralCrossRefGoogle Scholar
  92. 92.
    Jardine J, Julien J-P, Menis S, Ota T, Kalyuzhniy O, McGuire A, et al. Rational HIV immunogen design to target specific germline B cell receptors. Science. 2013;340(6133):711–6.PubMedPubMedCentralCrossRefGoogle Scholar
  93. 93.
    Cheng W, Curti E, Rezende WC, Kwityn C, Zhan B, Gillespie P, et al. Biophysical and formulation studies of the Schistosoma mansoni TSP-2 extracellular domain recombinant protein, a lead vaccine candidate antigen for intestinal schistosomiasis. Hum Vaccin Immunother. 2013;9(11):2351–61.PubMedPubMedCentralCrossRefGoogle Scholar
  94. 94.
    Alsenaidy MA, Okbazghi SZ, Kim JH, Joshi SB, Middaugh CR, Tolbert TJ, et al. Physical stability comparisons of IgG1-Fc variants: effects of N-glycosylation site occupancy and Asp/Gln residues at site Asn 297. J Pharm Sci. 2014;103(6):1613–27.PubMedPubMedCentralCrossRefGoogle Scholar
  95. 95.
    Qi W, Zeng Y, Orgel S, Francon A, Kim JH, Randolph TW, et al. Preformulation study of highly purified inactivated polio vaccine, serotype 3. J Pharm Sci. 2014;103(1):140–51.PubMedCrossRefPubMedCentralGoogle Scholar
  96. 96.
    Chen X, Choudhari SP, Martinez-Becerra FJ, Kim JH, Dickenson NE, Toth RT, et al. Impact of detergent on the biophysical properties and immune response of the IpaDB Fusion protein, a candidate subunit vaccine against Shigella spp. Infect Immun. 2015;83(1):292–9. Scholar
  97. 97.
    Kumru OS, Joshi SB, Thapa P, Pheasey N, Bullock PS, Bashiri H, et al. Characterization of an oncolytic herpes simplex virus drug candidate. J Pharm Sci. 2015;104(2):485–94.PubMedCrossRefPubMedCentralGoogle Scholar
  98. 98.
    Xia X, Kumru OS, Blaber SI, Middaugh CR, Li L, Ornitz DM, et al. Engineering a cysteine-free form of human fibroblast growth factor-1 for “second generation” therapeutic application. J Pharm Sci. 2016;105(4):1444–53.PubMedPubMedCentralCrossRefGoogle Scholar
  99. 99.
    Sahni N, Chaudhuri R, Hickey JM, Manikwar P, D’Souza A, Metters A, et al. Preformulation characterization, stabilization, and formulation design for the Acrylodan-Labeled Glucose-Binding Protein SM4-AC. J Pharm Sci. 2017;106(5):1197–210.PubMedCrossRefPubMedCentralGoogle Scholar
  100. 100.
    Toprani VM, Hickey JM, Sahni N, Toth RT IV, Robertson GA, Middaugh CR, et al. Structural characterization and physicochemical stability profile of a double mutant heat labile toxin protein based adjuvant. J Pharm Sci. 2017;106(12):3474–85.PubMedPubMedCentralCrossRefGoogle Scholar
  101. 101.
    Wei Y, Wahome N, Kumar P, Whitaker N, Picking WL, Middaugh CR. Effect of phosphate ion on the structure of lumazine synthase, an antigen presentation system from bacillus anthracis. J Pharm Sci. 2018;107(3):814–23.PubMedCrossRefPubMedCentralGoogle Scholar
  102. 102.
    Kramer RM, Archer MC, Orr MT, Cauwelaert ND, Beebe EA, Huang P-D, et al. Development of a thermostable nanoemulsion adjuvanted vaccine against tuberculosis using a design-of-experiments approach. Int J Nanomedicine. 2018;13:3689–711.PubMedPubMedCentralCrossRefGoogle Scholar
  103. 103.
    Klijn ME, Hubbuch J. Application of empirical phase diagrams for multidimensional data visualization of high-throughput microbatch crystallization experiments. J Pharm Sci. 2018;107:2063–9.PubMedCrossRefPubMedCentralGoogle Scholar
  104. 104.
    Ausar SF, Espina M, Brock J, Thyagarayapuran N, Repetto R, Khandke L, et al. High-throughput screening of stabilizers for respiratory syncytial virus: identification of stabilizers and their effects on the conformational thermostability of viral particles. Hum Vaccin. 2007;3(3):94–103.PubMedCrossRefPubMedCentralGoogle Scholar
  105. 105.
    Kissmann J, Ausar SF, Foubert TR, Brock J, Switzer MH, Detzi EJ, et al. Physical stabilization of norwalk virus-like particles. J Pharm Sci. 2008;97(10):4208–18.PubMedCrossRefPubMedCentralGoogle Scholar

Copyright information

© American Association of Pharmaceutical Scientists 2020

Authors and Affiliations

  • Yangjie Wei
    • 1
  • Sangeeta B. Joshi
    • 1
  • Akhilesh Bhambhani
    • 1
    • 2
  • Yuhong Zeng
    • 1
    • 3
  • Nicholas R. Larson
    • 1
  • Gang Hu
    • 1
  • Eric J. Deeds
    • 4
    • 5
  • C. Russell Middaugh
    • 1
    Email author
  1. 1.Department of Pharmaceutical ChemistryMacromolecule and Vaccine Stabilization Center, University of KansasLawrenceUSA
  2. 2.Merck & Co. Inc.West PointUSA
  3. 3.ReForm BiologicsWoburnUSA
  4. 4.Department of Molecular BiosciencesUniversity of KansasLawrenceUSA
  5. 5.Center for Computational BiologyUniversity of KansasLawrenceUSA

Personalised recommendations