Pharmaceutical Research

, Volume 22, Issue 1, pp 33–37 | Cite as

Concentration Determination of a Recombinant Vaccine Antigen Adsorbed onto an Alum Adjuvant by Chemiluminescent Nitrogen Detection

  • John V. Amari
  • Philip Levesque
  • Zhirui Lian
  • Trish Lowden
  • Uditha deAlwis

No Heading


A chemiluminescent nitrogen detector (CLND) has been evaluated for determining the concentration of an aluminum-adsorbed recombinant vaccine antigen.


Quantification of the antigen was based upon several nitrogen-containing compounds used to calibrate the CLND. All calibrants (6.75–400 μg/ml) generated linear standard curves, with slopes being directly proportional to the % nitrogen. The limit of quantification (LOQ) was determined to be 6.75 μg/ml based on the performance of the antigen standard curve, and the limit of detection (LOD) was defined by setting the CLND minimum peak area to 40,000 U. The CLND was capable of analyzing antigen-adjuvant suspensions (adsorbed + unbound antigen) without any sample pretreatment. To measure unbound antigen, the suspension was centrifuged and an aliquot of supernatant removed for analysis; the difference between these two measurements was the amount of adsorbed antigen.


The adjuvant exhibited no significant matrix effect. Samples were analyzed in triplicate with observed relative standard deviation values ranging from 0.065% to 10.0%. The most accurate concentrations of the antigen were recovered relative to the antigen itself and to glycine as standards.


This methodology provides a direct measurement of the concentration of a vaccine antigen adsorbed onto an aluminum adjuvant.

Key words:

adjuvant adsorbate vaccine alum chemiluminescent nitrogen detection nitrogen content 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    1. J. S. Kenney, B. W. Hughes, M. P. Masada, and A. C. Allison. Influence of adjuvants on the quality, affinity, isotype and epitope specificity of murine antibodies. J. Immunol. Methods 121: 157–166 (1989).Google Scholar
  2. 2.
    2. S. Shirodkar, R. L. Hutchinson, D. L. Perry, J. L. White, and S. L. Hem. Aluminum compounds used as adjuvants in vaccines. Pharm. Res. 7:1282–1288 (1990).Google Scholar
  3. 3.
    3. J. O. Naim, C. J. van Oss, W. Wu, R. F. Giese, and P. A. Nickerson. Mechanisms of adjuvancy: I-metal oxides as adjuvants. Vaccine 15:1183–1193 (1997).Google Scholar
  4. 4.
    4. G. S. Deepe Jr. Prospects for the development of fungal vaccines. Clinical Microbio Reviews 10:585–596 (1997). Google Scholar
  5. 5.
    5. L. S. Burrell, J. L. White, and S. L. Hem. Stability of aluminum-containing adjuvants during aging at room temperature. Vaccine 18:2188–2192 (2000).Google Scholar
  6. 6.
    6. R. W. Ellis. Technologies for the design, discovery, formulation and administration of vaccines. Vaccine 19:2681–2687 (2001).Google Scholar
  7. 7.
    7. R. W. Ellis. Product development plan for new vaccine technologies. Vaccine 19:1559–1566 (2001).Google Scholar
  8. 8.
    8. H. HogenEsch. Mechanisms of stimulation of the immune response by aluminum adjuvants. Vaccine 20:S34–S39 (2002).Google Scholar
  9. 9.
    9. W. Matheis, A. Zott, and M. Schwanig. The role of the adsorption process for production and control combined adsorbed vaccines. Vaccine 20:67–73 (2002).Google Scholar
  10. 10.
    10. M. Singh and D. O’Hagan. Advances in vaccine adjuvants. Nat. Biotechnol. 17:1075–1081 (1999).Google Scholar
  11. 11.
    11. R. H. Al-Shakhshir, F. E. Regnier, J. L. White, and S. L. Hem. Contribution of electrostatic and hydrophobic interactions to the adsorption of proteins by aluminum-containing adjuvants. Vaccine 13:41–44 (1995).Google Scholar
  12. 12.
    12. L. S. Burrell, E. B. Lindblad, J. L. White, and S. L. Hem. Stability of aluminum-containing adjuvants to autoclaving. Vaccine 17: 2599–2603 (1999).Google Scholar
  13. 13.
    13. J. V. Rinella Jr., R. F. Workman, M. A. Hermodson, J. L. White, and S. L. Hem. Elutability of proteins from aluminum-containing vaccine adjuvants by treatment with surfactants. J. Colloid Inter-face Sci. 197:48–56 (1998).Google Scholar
  14. 14.
    14. J. V. Rinella Jr., J. L. White, and S. L. Hem. Effect of pH on the elution of model antigens from aluminum-containing adjuvants. J. Colloid Interface Sci. 205:161–165 (1998).Google Scholar
  15. 15.
    15. J. M. Heimlich, F. E. Regnier, J. L. White, and S. L. Hem. The in vitro displacement of absorbed model antigens from aluminum-containing adjuvants by interstitial proteins. Vaccine 17:2873–2881 (1999).Google Scholar
  16. 16.
    16. N. E. Raya, M. M. Luaces, R. S. Rodriguez, C. N. Galvez, M. P. Rivero, N. M. de la Puente, M. F. Batista, and G. G. Nieto. Preformulation study of the vaccine candidate P64k against Neisseria meningitides. Biotechnol. Appl. Biochem. 29:113–117 (1999).Google Scholar
  17. 17.
    17. M. Chang, Y. Shi, and S. L. Nail, H. HogenEsch, S. B. Adams, J. L. White, and S. L. Hem. Degree of antigen adsorption in the vaccine or interstitial fluid and its effect on the antibody response in rabbits. Vaccine 19:2884–2889 (2001).Google Scholar
  18. 18.
    18. Y. Shi, H. HogenEsch, and S. L. Hem. Change in the degree of adsorption of proteins by aluminum-containing adjuvants following exposure to interstitial fluid: freshly prepared and aged model vaccines. Vaccine 20:80–85 (2002).Google Scholar
  19. 19.
    19. S. L. Hem. Elimination of aluminum adjuvants. Vaccine 20:S40–S43 (2002).Google Scholar
  20. 20.
    20. K. R. Aiyar, R. I. W. Greig, and M. Sangrouber. Nitrogen determination of commercially prepared lactalbumin. J. Food Sci. 51: 856–858 (1986).Google Scholar
  21. 21.
    21. D. L. Berner and J. Brown. Protein nitrogen combustion method collaborative study I. Comparison with Smally total Kjeldahl nitrogen and combustion results. JAOCS 71:1291–1293 (1994).Google Scholar
  22. 22.
    22. J. M. Lynch, D. M. Barbano, P. A. Healy, and J. R. Fleming. Performance evaluation of direct forced-air total solids and Kjeldahl total nitrogen methods: 1990 through 1995. J. Assoc. Off. Anal. Chem. Intl. 80:1038–1043 (1997).Google Scholar
  23. 23.
    23. J. M. Lynch and D. M. Barbano. Kjeldahl nitrogen analysis as a reference method for protein determination in dairy products. J. Assoc. Off. Anal. Chem. Intl. 82:1389–1398 (1999).Google Scholar
  24. 24.
    24. D. J. Levy, H. A. Bissell, and S. F. O’Keefe. Conversion of nitrogen to protein and amino acids in wild fruits. J. Chem. Ecol. 26:1749–1763 (2000).Google Scholar
  25. 25.
    25. C. L. Suard, R.-M. Mourel, D. Didenot, and M. H. Feinberg. Mechanisms involved in Kjeldahl microwave digestion of amino acids. J. Agric. Food Chem. 45:1202–1208 (1997).Google Scholar
  26. 26.
    26. E. M. Fujinari and J. D. Manes. Nitrogen-specific detection of peptides in liquid chromatography with a chemiluminescent nitrogen detector. J. Chromatgr. A 676:113–120 (1994).Google Scholar
  27. 27.
    27. E. M. Fujinari, J. D. Manes, and R. Bizanek. Peptide content determination of crude synthetic peptides by reversed-phase liquid chromatography and nitrogen-specific detection with a chemiluminescent nitrogen detector. J. Chromatgr. A 743:85–89 (1996).Google Scholar
  28. 28.
    28. E. M. Fujinari and J. D. Manes. Determination of molecular-mass distribution of food-grade protein hydrolyzates by size-exclusion chromatography and chemiluminescent nitrogen detection. J. Chromatgr. A 763:323–329 (1997).Google Scholar
  29. 29.
    29. A. Korner. Uncovering deficiencies in mass balance using HPLC with chemiluminescence nitrogen-specific detection. LC GC 20: 364–373 (2000).Google Scholar
  30. 30.
    30. E. W. Taylor, M. G. Qian, and G. D. Dollinger. Simultaneous on-line characterization of small organic molecules derived from combinatorial libraries for identity, quantity, and purity by reversed-phase HPLC with chemiluminescent nitrogen, UV, and mass spectrometric detection. Anal. Chem. 70:3339–3347 (1998).Google Scholar

Copyright information

© Springer Science+Business Media, Inc. 2005

Authors and Affiliations

  • John V. Amari
    • 1
    • 2
  • Philip Levesque
    • 1
  • Zhirui Lian
    • 1
  • Trish Lowden
    • 1
  • Uditha deAlwis
    • 1
  1. 1.ID Biochemical Corporation of NorthboroughNorthboroughUSA
  2. 2.Syntonix PharmaceuticalsWalthamUSA

Personalised recommendations