Electrospray ionization (ESI) has significantly increased the size of proteins and biopolymers that can be detected by most
mass spectrometers, and provides a means to accurately characterize component vaccines. This is accomplished by generating
multiple charge states by ESI and using deconvolution software to analyze the spectra. This technology added a separation
capability, through HPLC, to the analysis, allowing vaccine components to be separated online before mass spectrometric analysis.
With the advent of high-resolution mass spectrometers such as the Quadrupole–Time of Flight (Q–ToF) and highly sensitive MS
instruments, such as the Quadrupole–Ion Trap (Q–Trap), characterization of vaccines has entered a new phase. Minor modifications
to the immunogenic protein or nucleic acid can now be detected and a better understanding can be achieved of what causes vaccines
to fail as they age. These new techniques have not replaced older cell-based potency assays, however, because the mechanisms
connecting these structural modifications to functional differences have not been fully established. Many modifications have
no effect on the efficacy of the vaccine, but modifications to the sections of the protein or nucleic acid that interact with
the host immune system can change or eliminate the ability of the immune system to recognize the epitopes and thus elicit
an immune response. By combining these data with the results of potency and toxicity studies, a new picture may be developed
of the changes in the vaccines that cause these failures.
New advances in analytical technology have also provided a means to characterize these vaccines to a degree that had not previously
been possible. Older techniques such as gel electrophoresis (one and two dimensional) and affinity chromatography are being
augmented by high-resolution, high-accuracy mass spectrometric methods as well as the newly evolved field of protein NMR.
The mass spectrometric methods offer the ability to identify even very minor structural changes in the protein or lipid components
of the vaccine with a high degree of accuracy. The NMR methods provide powerful conformational data regarding biopolymers
present in the vaccines. These changes in the structure of vaccine components may have previously gone undetected using the
classical techniques of vaccine characterization.
High resolution mass spectrometry provides the ability to better characterize the changes in the immunogenic protein or nucleotide
that result in changes in physical properties. Q–ToF can provide detailed sequence information about these nucleotides and
proteins to a level that the physical techniques cannot. Through the use of enzymatic digestions, even large proteins can
be broken down into manageable fragments for sequencing. The peptide bonds between amino acids and the bonds in the phosphate
backbone of oligonucleotides are the weak points in the structures and fragment predictably during MS–MS analysis. The high-mass
accuracy of the mass spectrometers allows unambiguous assignment of sequences based on molecular weight data and can be combined
with MS–MS analysis when the molecular weight data provides more than one possibility. When dealing with known or simple vaccines,
this process is fairly straightforward.
When analyzing more complex vaccines, such as those containing complex adjuvants or multiple immunogenic components, the situation
becomes less clear. With these types of vaccines, the discriminatory power of the mass spectrometer is of great aid. HPLC–UV
analysis of these complex mixtures often results in multiple components co-eluting. Due to the heterogeneous composition of
these types of vaccines, this is often unavoidable. The mass spectrometer offers an additional level of separation through
its mass sensitive or selective detector. Even co-eluting components can be resolved through mass spectra.
These techniques have been successfully used, for example, to determine phospholipid composition and to characterize proteins
in complex vaccines. Phospholipids contain polar head groups that contain both phosphates, which respond strongly in negative
ion mode, and amine containing groups that respond well in positive ion mode electrospray. One common adjuvant is lecithin.
This material is composed of a number of related phospholipids that can be separated by HPLC and identified by their mass
spectra. Proteins such as the B subunit of cholera toxin can be characterized in much the same way as protein-based component
vaccines. Aluminum-based adjuvants can be detected through the use of ICP-MS, and other amine-based adjuvants are easily detected
through positive mode electrospray mass spectrometry. An overall fingerprint of a vaccine can be constructed using mass spectrometry
data combined with HPLC retention time data. This fingerprint can then be used to monitor changes in the vaccine from batch
to batch or as the vaccine ages, or to characterize reference standards during the process of standards development, evaluation,
and further improvement. This is the centerpiece in the field of developing effective (novel), affordable, and safe vaccine
formulations. These, in turn, will aid the global development and further refinement of good manufacturing practices for vaccines
and development of global policies and guidelines.