Small peptide antigens are normally conjugated to a large carrier protein to increase immunogenicity. The number of small
peptides per carrier protein in the conjugation product (i.e., the epitope density) is important for vaccine efficacy. Quantitation
methods for epitope density are needed for vaccine production control and stability determination. Amino acid analysis can
be used to determine the vaccine epitope density. After conjugating a small peptide to a larger carrier protein, the amino
acid composition will be changed. The epitope density can then be measured by comparing the amino acid compositions of the
unconjugated carrier protein and the vaccine peptide conjugated carrier protein. This method can only determine the mean epitope
density, not the distribution of the epitope density.
Mass spectrometry can be used to measure the epitope density by determining the molecular mass difference between the unconjugated
carrier protein and the vaccine peptide conjugated carrier protein. Both the mean and the distribution of epitope density
can be determined by mass spectrometry, but the method accuracy and precision are not as good as with amino acid analysis
due to the heterogeneous properties of the carrier protein and the conjugation. These two methodologies are complementary
to each other and it is recommended to use amino acid analysis to measure the mean epitope density and mass spectrometric
method to measure the epitope density distribution. The epitope density also can be measured with enzyme linked immunoassay
(ELISA) by using the antivaccine peptide antibody. Since the variation in ELISA method is normally significantly larger than
in physical-chemical methodology, this method is not commonly used to measure the epitope density. Instead, it is used to
measure the vaccine activity.
A summary of various vaccine characterization technologies are presented in Table 1. As can be seen, advanced mass spectrometry
as a tool has applications in all phases of vaccine development. The ability to characterize a vaccine to the levels now allowed
by modern mass spectrometers offers the potential for an unprecedented understanding of vaccines and why they fail. Classical
characterization techniques are often used as a screening method to identify which aged or stressed vaccines need further
investigation by mass spectrometry. All phases of vaccine development, from initial characterization to troubleshooting of
the manufacturing process, and all the way up to release testing of the final vaccine product, can benefit from these new
technologies. They may even be used to monitor the stability of vaccine materials once they have entered the marketplace.
It may eventually become possible to characterize the changes that make vaccines fail on a structural level, based on data
collected through the analysis of degraded vaccines by mass spectrometry. Mass spectrometry also provides a means to verify
the more classical techniques, and can be used as an adjunct to these methods to provide a deeper understanding of how vaccines
function This can lead to improvements that will make vaccines even safer and even more reliable and affordable.
Table 1. A summary of various vaccine characterization technologies
Xiaoya Ding, PhD, is the director of scientific and technical affairs at the cGMP Laboratory of PPD, 608.827.9400, Xiaoya.Ding@madison.ppdi.com
. At the same company, Steven Becht, PhD, is a research scientist and Xuelin Gu, PhD, is a laboratory manager.
1. US Food and Drug Administration. Guidance for Industry. Characterization and qualification of cell substrates and other
biological starting materials used in the production of viral vaccines for the prevention and treatment of infectious diseases.
Rockville, MD: 2006 Sept.
2. US FDA. Innovation stagnation: challenge and opportunity on the critical path to new medical products. Rockville, MD:
3. Word Health Organization. Drafting group on stability of vaccines guidelines: Report of the 2nd meeting. Geneva, Switzerland:
2004 June 16–17.
4. Goodman JL. Vaccine safety: vision for the future. Presentation at FDA vaccine safety workshop. 2007 Apr 10.
5. WHO Guidelines on Nonclinical Evaluation of Vaccines, WHO Technical Report Series, No. 927. Geneva: 2005.
6. Knezevic I. Potency Assays for Novel Vaccines: WHO Expectations. NIH meeting: Assaying potency of novel vaccines. 2005
Oct 11–12; Bethesda, MD.