Pharmaceutical Research

, 37:11 | Cite as

Design and Optimization of a Temperature-Stable Dry Powder BCG Vaccine

  • Dominique N. Price
  • Nitesh K. Kunda
  • Rajaun Ellis
  • Pavan MuttilEmail author
Research Paper



Loss of vaccine potency due to extreme temperature exposure during storage and transport remains a significant obstacle to the success of many vaccines, including the Bacille Calmette-Guérin (BCG) vaccine, the only vaccine available against Mycobacterium tuberculosis. BCG is a live, attenuated vaccine requiring refrigerated storage for viability. In this study, we formulated a temperature-stable BCG dry powder using the spray drying technique.


We employed a factorial design to optimize our formulation of stabilizing excipients that included L-leucine, bovine serum albumin, polyvinylpyrrolidone, mannitol, and trehalose. Powders were characterized for their particle size, yield, water retention and uptake, glass transition temperature, and aerosol performance. Three optimal powder carrier mixtures were selected from the factorial design for BCG incorporation based on their stability-promoting and powder flow characteristics. Vaccine powders were also assessed for BCG viability and in vivo immunogenicity after long-term storage.


Live BCG was successfully spray-dried using the optimized carriers. Dry powder BCG showed no loss in viability (25°C, up to 60% relative humidity; RH) and ~2-log loss in viability (40°C, 75% RH) after one year of storage. The aerodynamic size of the powders was in the respirable range. Further, when healthy mice were immunized intradermally with reconstituted BCG powders (storage for 2 years), the vaccine retained its immunogenicity.


We developed a spray-dried BCG vaccine that was viable and antigenic after long-term storage. To our knowledge, this is a first study to show room temperature stability of live BCG vaccine without any loss in viability for 12 months.

Key Words

BCG cold-chain factorial design live bacterial vaccine pulmonary vaccination spray drying tuberculosis vaccine formulation vaccine stability 


Acknowledgements and Disclosure

The authors would like to acknowledge Dr. Avni Patel for her help with the initial spray drying experiments. Laura Stephens for her help with the phagocytosis assay. We would also like to thank Dr. Hien Pham for the use of SEM, Dr. Eric Peterson for XRD, and Dr. Stephen Jett for TEM analysis.The authors declare no conflict of interest.


This work was funded in part by the Bill and Melinda Gates Grand Challenges Exploration (OPP1061393; PI- Dr. Pavan Muttil), National Institute of Allergy and Infectious Diseases (T32 Training Grant No: A1007538; Recipient- DNP), and by the National Research Foundation (Grant# 105830, South Africa; PI- Dr. Bernard Fourie).

Supplementary material

11095_2019_2739_MOESM1_ESM.docx (504 kb)
ESM 1 (DOCX 504 kb)


  1. 1.
    Persson S. Smallpox, syphilis and salvation: medical breakthroughs that changed the world [internet]. Exisle Publishing; 2010. Available:
  2. 2.
    Nabel GJ. Designing Tomorrow’s vaccines. N Engl J Med. 2013;368:551–60. Scholar
  3. 3.
    Wang G, Cao R-Y, Chen R, Mo L, Han J-F, Wang X, et al. Rational design of thermostable vaccines by engineered peptide-induced virus self-biomineralization under physiological conditions. Proc Natl Acad Sci U S A. 2013;110:7619–24. Scholar
  4. 4.
    Greenwood B. The contribution of vaccination to global health: past, present and future. Philos Trans R Soc Lond Ser B Biol Sci. 2014;369:20130433. Scholar
  5. 5.
    Matthias D, Robertson J, Garrison M, Newland S, Nelson C. Freezing temperatures in the vaccine cold chain: a systematic literature review. Vaccine. 2007;25:3980–6. Scholar
  6. 6.
    Black RE, Cousens S, Johnson HL, Lawn JE, Rudan I, Bassani DG, et al. Global, regional, and national causes of child mortality in 2008: a systematic analysis. Lancet Elsevier. 2010;375:1969–87. Scholar
  7. 7.
    Organization WH. Global Health Observatory data - immunization [internet]. World Health Organization; 2014. Available:
  8. 8.
    Galazka A, Milstien J, Zaffran M. Thermostability of vaccines. World Heal Organ Geneva. 1998; Available:
  9. 9.
    Cheyne J. Vaccine delivery management. Clin Infect Dis. 1989;11:S617–22. Scholar
  10. 10.
    Kaufmann JR, Miller R, Cheyne J. Vaccine supply chains need to be better funded and strengthened, or lives will be at risk. In: Health Affairs [Internet]. 2011; pp. 1113–1121. CrossRefGoogle Scholar
  11. 11.
    Lerman SJ. Measles in children previously vaccinated against measles. JAMA. 1971;216:1311. Scholar
  12. 12.
    Krugman RD, Meyer BC, Enterline JC, Parkman PD, Witte JJ, Meyer HM. Impotency of live-virus vaccines as a result of improper handling in clinical practice. J Pediatr. 1974;85:512–4. Scholar
  13. 13.
    World Health Organization (WHO). Temperature sensitivity of vaccines. 2006; 63. doi:WHO/IVB/06.10.Google Scholar
  14. 14.
    World Health Organization. Frequent exposure to suboptimal temperatures in vaccine cold-chain system in India: results of temperature monitoring in 10 states. World Health Organization; Available:
  15. 15.
    Zhang J, Pritchard E, Hu X, Valentin T, Panilaitis B, Omenetto FG, et al. Stabilization of vaccines and antibiotics in silk and eliminating the cold chain. Proc Natl Acad Sci U S A. 2012;109:11981–6. Scholar
  16. 16.
    Garcia-Contreras L, Wong Y-L, Muttil P, Padilla D, Sadoff J, Derousse J, et al. Immunization by a bacterial aerosol. Proc Natl Acad Sci U S A. 2008;105:4656–60. Scholar
  17. 17.
    Mansour HM, Xu Z, Hickey AJ. Dry Powder Aerosols Generated by Standardized Entrainment Tubes from Alternative Sugar Blends : 3 . Trehalose Dihydrate and D -Mannitol Carriers. J Pharm Sci. 2010;99:3430–41. Scholar
  18. 18.
    Amorij J-P, Huckriede A, Wilschut J, Frijlink HW, Hinrichs WLJ. Development of stable influenza vaccine powder formulations: challenges and possibilities. Pharm Res. 2008;25:1256–73. Scholar
  19. 19.
    Jin TH, Tsao E, Goudsmit J, Dheenadhayalan V, Sadoff J. Stabilizing formulations for inhalable powders of an adenovirus 35-vectored tuberculosis (TB) vaccine (AERAS-402). Vaccine Elsevier Ltd. 2010;28:4369–75. Scholar
  20. 20.
    Corbanie EA, Remon JP, Van Reeth K, Landman WJM, van Eck JHH, Vervaet C. Spray drying of an attenuated live Newcastle disease vaccine virus intended for respiratory mass vaccination of poultry. Vaccine. 2007;25:8306–17. Scholar
  21. 21.
    Sou T, Kaminskas LM, Nguyen T-H, Carlberg R, McIntosh MP, Morton DAV. The effect of amino acid excipients on morphology and solid-state properties of multi-component spray-dried formulations for pulmonary delivery of biomacromolecules. Eur J Pharm Biopharm. 2012;83:234–43. Scholar
  22. 22.
    Vehring R. Pharmaceutical particle engineering via spray drying. Pharm Res. 2008;25:999–1022. Scholar
  23. 23.
    Kunda NK, Wafula D, Tram M, Wu TH, Muttil P. A stable live bacterial vaccine. Eur J Pharm Biopharm. 2016;103:109–17. Scholar
  24. 24.
    Saboo S, Tumban E, Peabody J, Wafula D, Peabody DS, Chackerian B, et al. Optimized formulation of a Thermostable spray-dried virus-like particle vaccine against human papillomavirus. Mol Pharm. 2016;13:1646–55. Scholar
  25. 25.
    McAdams D, Chen D, Kristensen D. Spray drying and vaccine stabilization. Expert Rev Vaccines. 2012;11:1211–9. Scholar
  26. 26.
    Sou T, Meeusen EN, de Veer M, Morton DAV, Kaminskas LM, McIntosh MP. New developments in dry powder pulmonary vaccine delivery. Trends Biotechnol. 2011;29:191–8. Scholar
  27. 27.
    Geller DE, Weers J, Heuerding S. Development of an inhaled dry-powder formulation of tobramycin using PulmoSphere™ technology. J Aerosol Med Pulm Drug Deliv. 2011;24:175–82. Scholar
  28. 28.
    Dharmadhikari AS, Kabadi M, Gerety B, Hickey AJ, Fourie PB, Nardell E. Phase I, single-dose, dose-escalating study of inhaled dry powder capreomycin: a new approach to therapy of drug-resistant tuberculosis. Antimicrob Agents Chemother. 2013;57:2613–9. Scholar
  29. 29.
    Muttil P, Kaur J, Kumar K, Yadav AB, Sharma R, Misra A. Inhalable microparticles containing large payload of anti-tuberculosis drugs. Eur J Pharm Sci. 2007;32:140–50. Scholar
  30. 30.
    Zwerling A, Pai M. The BCG world atlas: a new, open-access resource for clinicians and researchers. Expert Rev Anti-Infect Ther. 2011;9:559–61. Scholar
  31. 31.
    Andersen P, Doherty TM. The success and failure of BCG - implications for a novel tuberculosis vaccine. Nat Rev Microbiol. 2005;3:656–62. Scholar
  32. 32.
    McShane H. Understanding BCG is the key to improving it. Clin Infect Dis. 2013;58:481–2. Scholar
  33. 33.
    Shur J, Nevell TG, Ewen RJ, Price R, Smith A, Barbu E, et al. Cospray-dried unfractionated heparin with L-leucine as a dry powder inhaler mucolytic for cystic fibrosis therapy. J Pharm Sci. 2008;97:4857–68. Scholar
  34. 34.
    Ohrem HL, Schornick E, Kalivoda A, Ognibene R. Why is mannitol becoming more and more popular as a pharmaceutical excipient in solid dosage forms? Pharm Dev Technol. 2014;19:257–62. Scholar
  35. 35.
    Gonnissen Y, Remon JP, Vervaet C. Development of directly compressible powders via co-spray drying. Eur J Pharm Biopharm. 2007;67:220–6. Scholar
  36. 36.
    Gonnissen Y, Remon JP, Vervaet C. Effect of maltodextrin and superdisintegrant in directly compressible powder mixtures prepared via co-spray drying. Eur J Pharm Biopharm. 2008;68:277–82. Scholar
  37. 37.
    Liao Y-H, Brown MB, Nazir T, Quader A, Martin GP. Effects of sucrose and trehalose on the preservation of the native structure of spray-dried lysozyme. Pharm Res. 2002;19:1847–53 Available: Scholar
  38. 38.
    Nguyen XC, Herberger JD, Burke PA. Protein powders for encapsulation: a comparison of spray-freeze drying and spray drying of darbepoetin alfa. Pharm Res. 2004;21:507–14. Scholar
  39. 39.
    Garmise RJ, Staats HF, Hickey AJ. Novel dry powder preparations of whole inactivated influenza virus for nasal vaccination. AAPS PharmSciTech. 2007;8:E81. Scholar
  40. 40.
    Maury M, Murphy K, Kumar S, Mauerer A, Lee G. Spray-drying of proteins: effects of sorbitol and trehalose on aggregation and FT-IR amide I spectrum of an immunoglobulin G. Eur J Pharm Biopharm. 2005;59:251–61. Scholar
  41. 41.
    Adler M, Lee G. Stability and surface activity of lactate dehydrogenase in spray-dried trehalose. J Pharm Sci. 1999;88:199–208. Scholar
  42. 42.
    Andya JD, Maa YF, Costantino HR, Nguyen PA, Dasovich N, Sweeney TD, et al. The effect of formulation excipients on protein stability and aerosol performance of spray-dried powders of a recombinant humanized anti-IgE monoclonal antibody. Pharm Res. 1999;16:350–8 Available: Scholar
  43. 43.
    Peyre M, Audran R, Estevez F, Corradin G, Gander B, Sesardic D, et al. Childhood and malaria vaccines combined in biodegradable microspheres produce immunity with synergistic interactions. J Control Release. 2004;99:345–55. Scholar
  44. 44.
    Johansen P, Men Y, Audran R, Corradin G, Merkle HP, Gander B. Improving stability and release kinetics of microencapsulated tetanus toxoid by co-encapsulation of additives. Pharm Res. 1998;15:1103–10 Available: Scholar
  45. 45.
    Dani B, Platz R, Tzannis ST. High concentration formulation feasibility of human immunoglubulin G for subcutaneous administration. J Pharm Sci. 2007;96:1504–17. Scholar
  46. 46.
    Liao Y-H, Brown MB, Jones SA, Nazir T, Martin GP. The effects of polyvinyl alcohol on the in vitro stability and delivery of spray-dried protein particles from surfactant-free HFA 134a-based pressurised metered dose inhalers. Int J Pharm. 2005;304:29–39. Scholar
  47. 47.
    Zeng XM, Martin GP, Marriott C. Effects of molecular weight of polyvinylpyrrolidone on the glass transition and crystallization of co-lyophilized sucrose. Int J Pharm. 2001;218:63–73. Scholar
  48. 48.
    Mahlin D, Berggren J, Gelius U, Engström S, Alderborn G. The influence of PVP incorporation on moisture-induced surface crystallization of amorphous spray-dried lactose particles. Int J Pharm. 2006;321:78–85. Scholar
  49. 49.
    Hawe A, Friess W. Physico-chemical lyophilization behavior of mannitol, human serum albumin formulations. Eur J Pharm Sci. 2006;28:224–32. Scholar
  50. 50.
    Bosquillon C, Rouxhet PG, Ahimou F, Simon D, Culot C, Préat V, et al. Aerosolization properties, surface composition and physical state of spray-dried protein powders. J Control Release. 2004;99:357–67. Scholar
  51. 51.
    Wong Y-L, Sampson S, Germishuizen WA, Goonesekera S, Caponetti G, Sadoff J, et al. Drying a tuberculosis vaccine without freezing. Proc Natl Acad Sci U S A. 2007;104:2591–5. Scholar
  52. 52.
    National Research Council (US) Committee. Guide for the Care and Use of Laboratory Animals [Internet]. National Academies Press (US); 2011. Available:
  53. 53.
    Price DN, Kusewitt DF, Lino CA, McBride AA, Muttil P. Oral tolerance to environmental mycobacteria interferes with intradermal, but not pulmonary, immunization against tuberculosis. PLOS Pathog Public Libr Sci. 2016;12:e1005614. Scholar
  54. 54.
    ICH. Stability testing of new drug substances and products Q1A(R2). Int Conf Harmon. 2003; 24. CrossRefGoogle Scholar
  55. 55.
    ICH. Q5C Stability Testing of Biotechnological/Biological Products. ICH Harmon Tripart Guidel. 1995; 1–8.Google Scholar
  56. 56.
    World Health Organization. Vaccine Handling [Internet]. Available:
  57. 57.
    Schaltz-Buchholzer F, Frankel HN, Benn CS. The real-life number of neonatal doses of Bacille Calmette-Guérin vaccine in a 20-dose vial. Glob Health Action Taylor Francis. 2017;10:1–4. Scholar
  58. 58.
    Saluja V, Amorij J-P, Kapteyn JC, de Boer AH, Frijlink HW, Hinrichs WLJ. A comparison between spray drying and spray freeze drying to produce an influenza subunit vaccine powder for inhalation. J Control Release. Elsevier B.V. 2010;144:127–33. Scholar
  59. 59.
    Lu D, Garcia-Contreras L, Muttil P, Padilla D, Xu D, Liu J, et al. Pulmonary immunization using antigen 85-B polymeric microparticles to boost tuberculosis immunity. AAPS J Springer N Y. 2010;12:338–47. Scholar
  60. 60.
    Mensink MA, Frijlink HW, van der Voort MK, Hinrichs WLJ. How sugars protect proteins in the solid state and during drying (review): mechanisms of stabilization in relation to stress conditions. Eur J Pharm Biopharm Elsevier. 2017;114:288–95. Scholar
  61. 61.
    Duddu SP, Dal Monte PR. Effect of glass transition temperature on the stability of lyophilized formulations containing a chimeric therapeutic monoclonal antibody. Pharm Res. 1997;14:591–5 Available: Scholar
  62. 62.
    Izutsu K, Yoshioka S, Terao T. Effect of mannitol crystallinity on the stabilization of enzymes during freeze-drying. Chem Pharm Bull (Tokyo). 1994;42:5–8 Available: Scholar
  63. 63.
    Costantino HR, Andya JD, Nguyen PA, Dasovich N, Sweeney TD, Shire SJ, et al. Effect of Mannitol crystallization on the stability and aerosol performance of a spray-dried pharmaceutical protein, recombinant humanized anti-IgE monoclonal antibody. J Pharm Sci. 1998;87:1406–11. Scholar
  64. 64.
    Leung SSY, Parumasivam T, Gao FG, Carter EA, Carrigy NB, Vehring R, et al. Effects of storage conditions on the stability of spray dried, inhalable bacteriophage powders. Int J Pharm NIH Public Access. 2017;521:141–9. Scholar
  65. 65.
    Zipursky S, Djingarey MH, Lodjo J-C, Olodo L, Tiendrebeogo S, Ronveaux O. Benefits of using vaccines out of the cold chain: delivering meningitis a vaccine in a controlled temperature chain during the mass immunization campaign in Benin. Vaccine. 2014;32:1431–5. Scholar
  66. 66.
    Kahn A-L, Kristensen D, Rao R. Extending supply chains and improving immunization coverage and equity through controlled temperature chain use of vaccines. Vaccine. 2017;35:2214–6. Scholar
  67. 67.
    Chang BS, Mahoney RR. Enzyme thermostabilization by bovine serum albumin and other proteins: evidence for hydrophobic interactions. Biotechnol Appl Biochem. 1995;22:203–14 Available: Scholar
  68. 68.
    Price DN, Muttil P. Directed Intervention and Immunomodulation against Pulmonary Tuberculosis. In: Torchilin V, editor. Drug Delivery Systems for Tuberculosis Prevention and Treatment. Chichester: John Wiley & Sons, Ltd; 2016. p. 346–77. Scholar
  69. 69.
    Ernst JD. The immunological life cycle of tuberculosis. Nat Rev Immunol. Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. 2012;12:581–91. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Dominique N. Price
    • 1
    • 2
  • Nitesh K. Kunda
    • 1
    • 3
  • Rajaun Ellis
    • 4
    • 5
  • Pavan Muttil
    • 1
    Email author
  1. 1.Department of Pharmaceutical Sciences, College of PharmacyUniversity of New Mexico, Health Sciences CenterAlbuquerqueUSA
  2. 2.Biomedical Sciences Graduate ProgramUniversity of New Mexico, Health Sciences CenterAlbuquerqueUSA
  3. 3.Department of Pharmaceutical Sciences, College of Pharmacy and Health SciencesSt. John’s UniversityJamaicaUSA
  4. 4.Health Sciences CenterUniversity of New MexicoAlbuquerqueUSA
  5. 5.Nova Southeastern UniversityFort LauderdaleUSA

Personalised recommendations