CHALLENGES IN ANALYTICAL CHARACTERIZATION
Table I: Advantages and disadvantages of approaches used in vaccine characterization. CD is circular dichroism, and FTIR
is Fourier transform infrared spectroscopy.
|
Development of safe, effective and affordable vaccines requires that along with the use of suitable manufacturing approaches
there is effective testing of manufacturing intermediates and an application of modern characterization approaches (see Table
I). The difficulty of characterizing complex biological products such as vaccines makes it especially challenging to ensure
that they can be manufactured in a consistent, reproducible, and commercially viable manner with assurance of safety, quality,
and efficacy. The risk of manufacturing inconsistencies is especially high for novel products, because traditional testing
technologies might not be able to identify subtle and unanticipated variability (7).
Analytical testing of vaccines, just as any other pharmaceutical product, provides evidence that the vaccine and its intermediates
meet the specifications defined within the license application. Safety, efficacy, and potency tests associated with a licensed
vaccine are maintained within the approved filing and published in 21 CFR Part 610 in the US. In addition, several pharmacopeias (e.g., the Indian Pharmacopeia, British Pharmacopeia, US Pharmacopeia,
and European Pharmacopeia) publish monographs for vaccines to provide standardized requirements for commercialization. Most
countries require that vaccines be tested for both safety and efficacy by the manufacturer and a national testing laboratory
(e.g., the FDA Center for Biologics Evaluation and Research in the US) before release and distribution.
Because of their simple composition (i.e., a few well defined immunogenic molecules plus adjuvant), component vaccines are
the most amenable to analytical characterization. Live or killed/attenuated vaccines usually are a complex mixture of immunogens
since they are directly derived from organisms such as killed or attenuated virus, intact bacteria, or multiple bacterial
components. For such vaccines, the biological matrix is rather complex, allowing more characterization to be focused on the
adjuvant. Technological advances such as proteomics are likely to permit the characterization of the biological components
of such vaccines going forward. In the case of live and attenuated vaccine material, for example Bacillus Calmette-Guerin
vaccine and oral polio vaccine, the efficacy of each vaccine batch is related to the number of live particles determined either
by counting or by titration, that is, entirely in vitro. In vivo testing is only carried out for a new seed strain. Unlike live vaccines that are quantified by in vitro titration, an in vivo potency test is required for each batch of inactivated vaccines, although some exceptions do remain (8).
Classical methods for characterization of vaccines
These methods rely on the study of physical-chemical parameters such as differential scanning calorimetry (DSC), thermo-gravimetric
analysis (TGA), pH, protein content determination, elemental composition, and studying the effects of stress such as freeze-thaw
and agitation (9). TGA and DSC can be used to assess a change in the denaturing point of the protein or polynucleotide present.
Appearance and pH can be used to monitor changes in composition or the impact of stress as observed by clumping or discoloration.
Particle size analysis and particle size distribution can provide further insight into clumping and exposure to stressful
conditions which can significantly affect the safety and efficacy of the vaccine. However, these methods are unable to determine
small changes or relate the change in any of the measured parameters to an effect on potency and safety of the vaccine.
Advanced approaches to vaccine characterization
Lately, mass spectrophotometer (MS) based approaches have been applied towards product characterization as well as for routine
monitoring during commercial manufacturing. These techniques include inductively coupled plasma MS (ICP–MS), gas chromatography
MS (GC–MS), high performance liquid chromatography MS (HPLC–MS) and electrospray ionization time of flight MS (ESI–QToF–MS).
ICP–MS can be used to characterize a vaccine preparation by measuring concentrations of heavy metals which may have been introduced
unintentionally. HPLC–MS and GC–MS can measure the molecular weight of the vaccine components. To determine the exact location
of any changes in molecular structure, a MS–MS instrument can be used because the fragmentation events for these types of
molecules occur in a predictable manner that allow the interpretation of even complex spectra. ESI–MS has significantly increased
the range of the size of molecules whose mass can be accurately measured and therefore provides a means for characterization
of component based vaccines. In addition, newer MS-based instruments such as Q–TOF and Q–Trap have further improved sensitivity
to where MS based measurements can provide detailed understanding of structural changes in an immunogen which can be correlated
to the potency of an antigen.
The fact that analytical characterization plays a very crucial role in maintaining the potency and efficacy of a vaccine during
purification and processing is evident from the Hepatitis B vaccine characterization reported by Seo et al. (10). They performed
N-terminus sequencing of both monomers and dimers formed by complete and partial reduction, respectively, of the S-HBVsAg particles
under reducing SDS–PAGE condition. They demonstrated that each polypeptide within a S-HBVsAg particle has an authentic sequence
of N-terminus. Furthermore, a denaturation plot showed that the S-HBVsAg vaccine particles were extremely stable, especially in
solutions with high acidity. Such information is not only important for formulation purposes, but also provides insight into
appropriate conditions to be applied during downstream processing.
Another application highlighting use of advanced tools for characterization of vaccine products was used by researchers at
FDA (11). Using NMR as a microscope to study polysaccharides at the molecular and atomic levels, the researchers probed the
individual atoms and their locations in relationship to each other. This information helped determine the molecular shapes
of these molecules, which in turn provided valuable insights into how polysaccharides interact with antibodies and proteins.
Tools such as laser light scattering and circular dichroism were suggested to characterize the overall size and shape of polysaccharides,
which, together with NMR, enable FDA and the pharmaceutical industry to ensure that polysaccharide vaccines meet regulatory
requirements for safety and effectiveness.
Table II: Diverse methods suitable for vaccine characterization listed based on the type of vaccine under evaluation. ELISA
is enzyme-linked immunosorbent assay, NMR is nuclear magnetic resonance, MS is mass spectroscopy, and HPLC is high-performance
liquid chromatography.
|
It is clear that the relevance of an analytical method with respect to its usefulness in characterizing an antigen depends
on the type of material (see Table II). A set of methods need to be employed to gain insight into the quality of a vaccine.
Production of well-characterized vaccines paves the path for reduced animal testing while providing safe and potent vaccines
to patients and is a path that must be pursued.
|
