Key Considerations for Development and Production of Vaccine Products - Challenges of vaccine development include regulatory, technical, and manufacturing hurdles in translating a vaccine candidate in

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Key Considerations for Development and Production of Vaccine Products
Challenges of vaccine development include regulatory, technical, and manufacturing hurdles in translating a vaccine candidate into a commercial product.


BioPharm International Supplements
Volume 25, Issue 3, pp. s28-s34

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.


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