Vaccine Characterization Using Classical Methods
The classical methods of vaccine characterization rely on the study of physical-chemical properties using methods such as
differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), pH, various stress conditions (agitation, freeze-thaw,
etc.) based on particulate formation, and methods of quantitating protein content as well as elemental composition. While
these methods are capable of determining whether or not the end product is consistent with previous batches, they are unable
to detect small changes that can result in a vaccine with reduced or even lost preventative characteristics. Not all changes
to the structure of the vaccine components have physical consequences, but many of them result in reduced vaccine performance.
Most of these techniques lack sensitivity when it comes to detecting small changes in the structure of the vaccine components
that can cause them to fail during use. Some changes can cause severe side reactions even in small quantities.
TGA and DSC are used to analyze the denaturing point of the vaccine protein or nucleotide. These tests generally give indirect
indications of changes in the vaccine with time and stress. Changes in protein sequence or modifications can substantially
affect denaturing kinetics, but these techniques provide no way to correlate these changes with actual changes in the structure
of the molecule. Appearance and pH are used to monitor major changes in the composition of the vaccines and are relatively
insensitive to these changes. Other physical characteristics that affect vaccine function include particle size and particle
size distribution. Clumping of the vaccine antigen can degrade the function of the vaccine and can cause unwanted side effects.
Specific tests for quantitating proteins or oligonucleotides, such as elemental analysis and total protein content (bicinchoninic
acid, or BCA) tests, can provide vital quality control data for troubleshooting manufacturing problems, but they are of limited
value in analyzing degradation of the vaccines since elemental composition changes from degradants represent only a small
percentage of the overall elemental composition. In addition, most protein degradants will still be identified as proteins
in a total protein analysis. The conditions used for these assays also break up clumped proteins or oligonucleotides and are
insensitive to most changes caused by minor structural modifications of these molecules.
Vaccine Characterization Using Mass Spectrometry Technologies
Various mass spectrometry (MS) technologies are readily available today for use in vaccine characterization to assist in understanding
vaccine properties and functions for all phases of vaccine development processes from vaccine discovery, development, formulation,
manufacturing, stability, QC, and release, to post-market surveillance. ICPMS, GC–MS, HPLC–MS, and ESI–Q-ToF–MS are a few
of the MS technologies that can be used extensively in vaccine characterization.
ICP–MS is used to qualitatively and quantitatively measure toxic metals that can be introduced into a vaccine by the manufacturing
process and can have severe negative effects when the vaccine goes into widespread use. The ICP–MS technique oxidizes all
of the organic components in the vaccine to CO2, leaving behind all of the metal ions to be analyzed by either hot or cold plasma mass spectrometry. The results obtained
from ICP-MS analysis can provide some insights into loss of metal co-factors and other groups.
GC–MS and HPLC–MS are used to determine the molecular weight of the various vaccine components. These technologies can provide
a means of determining if a change in the molecular structure has occurred, but generally cannot localize it. Improvements
in the sensitivity of mass spectrometers and the increasing mass accuracy of the instruments have improved the capabilities
of these tests to the point where they can identify even minor changes in the vaccine components. Localizing these modifications
relies on another feature of the mass spectrometer. By placing a collision cell between two mass analyzers, additional structural
information can be obtained. In the case of proteins and oligonucleotides, these fragmentation events occur in a predictable
manner that allows interpretation of even very complex spectra. With instruments possessing a high mass accuracy, localization
of modifications to the protein can be accomplished. Many software packages currently available can automate this process