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Rianne Roukema is a corporate communications manager at OctoPlus NV
Bas Kremer, PhD, is a scientist in vaccine development at OctoPlus NV.
Leo de Leede is a director of preclinical research and development at OctoPlus NV.
The disadvantages of the traditional vaccine regime (prime plus boost) have spurred the development of single-shot vaccines. This article describes the development and manufacture of a prototype single-shot vaccine that uses microspheres made from cross-linked modified dextran polymers for controlled release of the antigen.
To provide effective patient protection, many traditional vaccines require multiple injections, which results in a costly and inconvenient regimen. These disadvantages have spurred the development of single-shot vaccines that can provide protection against infection with only one injection. This article describes the manufacture and early results of a prototype single-shot vaccine combining a prime and booster dose in one injection. The development process is based on the example of a hepatitis B vaccine.
(STEVE COLE/GETTY IMAGES)
Vaccines contribute significantly to improving world health by prevent the debilitating, and in some cases, fatal effects of infectious diseases by inducing a protective immune response against the causative agent. Vaccines' success is limited, however, by the need for multiple injections, and because this costly and inconvenient regimen often leads to logistical challenges and poor patient compliance. Recent advances in the use of biodegradable microsphere-based systems show the potential of developing single-shot vaccines to overcome this limitation. Based on the example of hepatitis B vaccination, this article describes the development process of a prototype single-shot vaccine combining a prime and booster dose in one injection.
Figure 1. Illustration of single-shot (B) versus traditional (A) vaccination schemes
The single-shot vaccine is a combination product of a prime component—antigen with an appropriate adjuvant—and a microsphere component that encapsulates antigen and provides the booster immunizations by delayed release of the antigen (Figure 1). Many aspects need to be taken into consideration when developing such controlled release technology-based vaccines (Table 1).
Table 1. Important determinants for single-shot vaccine development
The microsphere component uses OctoPlus's proprietary OctoVAX microsphere technology, which is based on cross-linked modified dextran polymers. Dextrans are ideal polymers to form biocompatible hydrogels. Two major advantages of dextran microspheres as protein delivery systems are that the particles are prepared in the absence of organic solvents, and that degradation of the microspheres does not result in a pH drop. Both exposure to organic solvents and an acidic environment are known to negatively affect protein stability.1 Several different dextrans have been developed for hydrogel formation. One of these dextran-based polymers is derivatized with hydroxy-ethyl methacrylate (dex-HEMA, Figure 2), which introduces hydrolytically sensitive carbonate ester groups that ensure biodegradation under physiological conditions.2 Studies have shown that protein therapeutics developed with this polymer retain the activity of the encapsulated protein following encapsulation and release.3
Figure 2. Chemical structure of dex-HEMA, the building block of the hydrogel microspheres. The carbonate ester site that confers hydrolytic sensitivity to the dex-HEMA is indicated.
Important factors in the manufacture of a microsphere-based vaccine are high encapsulation efficiency and a consistent particle-production process. Several formulation parameters play an important role in obtaining a robust process. Below, we discuss the processes and equipment used to manufacture several formulations.
Figure 3. Schematic representation of the microsphere preparation process
Dex-HEMA has been shown to be very suitable for the formation of the hydrogel that facilitates controlled release of encapsulated proteins. A microsphere formulation process has been developed based on this polymer (Figure 3).4 In this process, an emulsion of aqueous dex-HEMA solution is formed in an aqueous polyethylene glycol (PEG) solution, by mixing them in a bioreactor vessel (Figure 4). To ensure consistently high encapsulation efficiencies, the protein to be encapsulated is added to the dex-HEMA solution before adding the PEG solution. Subsequently, microspheres are obtained by polymerizing the HEMA groups using potassium persulfate (KPS) as initiator and N,N,N',N'-tetramethylethylenendiamine (TEMED) as the catalyst. After extensive washing, the final microsphere suspension can be filled into vials and freeze-dried to stabilize the product.
Figure 4. Bioreactor used during scale-up of the production process
Several factors are critical parameters for the formulation of consistent microspheres. First, the size distribution of the microspheres can be controlled by the shear force applied during the emulsification step in the bioreactor vessel. Factors that have been identified to influence this shear force are the mechanical stirring speed in the bioreactor vessel and the viscosity of the PEG solution, which is determined by the concentration and molecular weight of the PEG. Second, the presence of excipients in the starting composition can influence the matrix density and encapsulation efficiency of the microsphere product, either by a direct effect on the microsphere formation or on the protein characteristics.5 Finally, polymerization conditions such as KPS concentration, pH, and temperature, can influence the strength of the formed hydrogel matrix.6
The dextran microsphere preparation method, described by Stenekes et al., 4 was initially performed on a 5-g scale (containing 120 mg of microspheres), and used vortexing as a means to emulsify the dex-HEMA phase in the continuous PEG phase. However, vortexing is not practical at large scale. Therefore, we evaluated the feasibility of stirring, a process that is relatively easy to scale up, as a means of emulsification, ultimately at a 500-g scale.
A direct correlation was observed between stirring speed and mean particle diameter of the microspheres (Figure 5), thus confirming that the particle size of the dextran emulsion is dependent on the energy input during emulsification. It is important to note that, despite the larger mean diameter of microspheres prepared at the 500-g scale, more than 90% of the resulting particles had a size below 90 μm, a size suitable for subcutaneous injection.
Figure 5. Microscopy pictures of microspheres produced at a high stirring speed of 700 rpm (A) or a low stirring speed of 60 rpm (B). Pictures were taken at 1,000 x magnification; bars indicate 30 Î¼m.
To optimize the stirring process, the production set-up was transferred from regular laboratory equipment and glassware to an autoclavable 2-L jacketed bioreactor unit equipped with baffles and a stirring assembly. The production process has now been scaled up to 1,500 g with a production of miscrospheres averaging 40 μm in size.
Once the freeze-dried microsphere product is rehydrated by reconstitution in an aqueous solution, hydrolysis of the carbonate ester groups in the dex-HEMA will be initiated. This will increase the mesh size in the hydrogel network. The encapsulated protein will be released when the mesh size exceeds the hydrodynamic diameter of the protein. The actual release profile of the encapsulated protein is primarily determined by the matrix density.
It has been shown that the release of proteins and liposomes can be tailored from days to months by varying the main factors influencing the matrix density, which are the relative number of HEMA groups in dex-HEMA (degree of substitution, DS) and the initial water content of the microspheres.2,4,7,8 In Figure 6, the effects of different DS and initial water content on in vitro release profiles of a model protein, IgG, are presented.
Figure 6. The effect of dex-HEMA DS and initial water content on in vitro release of IgG. In vitro release of proteins is measured in time during incubation at physiological conditions. Release is expressed as percent of the maximal released amount of protein.
Most other microsphere delivery technologies, such as the poly-lactic-co-glycolic acid (PLGA)–based delivery technology, comprise process steps that expose the protein to potentially detrimental organic solvents. In contrast, the OctoVAX microsphere formulation is a completely aqueous process, which limits the effects of manufacturing on the integrity of the encapsulated proteins. Furthermore, because of the open matrix of the microspheres, hydrolytic degradation does not induce local acidification that may affect the protein's activity. As part of our single-shot vaccine development program, we have recently shown in a mouse model that the potency to induce an antibody response is comparable between encapsulated HBsAg and freely injected HBsAg (Figure 7). These data suggest that the HBsAg particle domains that induce these antibody responses are not affected by the formulation process or by the release process.
Figure 7. Antibody titers induced by HBsAg administered as a protein solution, admixed with placebo microspheres (MSP), or encapsulated in OctoVAX microspheres (MSP). Antibody titers at day 84 are represented as geometric mean titers per group of 10 animals in relation to an international reference standard.
To show the potential of the single-shot approach, prototypes were tested in in vivo studies. The results reveal the impact of the composition of the formulation on the immune response.
The OctoVAX microsphere technology is applied with the objective to reduce the number of injections to a single-shot vaccination that confers full protection against a specific infection for a period at least similar to the conventional regimen. For proof of concept of the single-shot vaccine approach, prototype vaccines were developed using the HBsAg antigen. Current practice for hepatitis B vaccination is either a two- or a three-injection regimen, consisting usually of a prime injection followed by two booster injections at 1–2 months and 3–12 months after the first administration. This vaccine consists of aluminum-adjuvanted HBsAg.
Based on the in vitro release profiles, prototype microsphere formulations were selected for further investigation of the concept. Proof of concept studies were performed by immunizing Balb/c mice with prototype microsphere vaccine formulations that combine a prime dose with a single microsphere component. This microsphere component represents the booster dose with a delayed antigen release around day 30. In these studies, the prototype single-shot vaccines were compared with two injections at day 0 and 28, representing the prime and booster dose. The potency of the vaccines to induce an antibody immune response was determined by analyzing the development, over time, of serum anti-HBsAg immunoglobulin concentration using a commercial immunoassay.
The results of these animal studies show that the single-shot HBsAg vaccines based on the OctoVAX controlled release technology induce responses that are similar to those induced by the representative two-injection control vaccination by subcutaneous administration. Figure 8 shows that vaccines with differences in physical aspects and adjuvant presence induce antibody responses with different kinetics. Formulation A induced the desired levels of antibodies without a prime component, in contrast to formulation B, which needed an adjuvanted prime. However, even with formulation B, the data show that OctoVAX microspheres can provide a booster dose on initiation of an immune response by a prime component, providing proof of concept for the single-shot controlled delivery principle. In additional experiments, it has been shown that intramuscular application results in similar or better immune responses, which may ultimately be the preferential choice for route of administration (data not yet published).
Figure 8. Antibody titers induced by prototype single-shot HBsAg vaccines based on microspheres of formulation A or B, which differ in their physical characteristics. Microspheres were either administered by themselves or mixed with aluminum hydroxide (AlOH)-adjuvanted HBsAg. Control mice received two injections of AlOH-adjuvanted HBsAg. Antibody titers are represented as geometric mean titers per group of 10 animals in relation to an international reference standard at 28, 56, and 84 days after injection.
In addition to the efficacy data, animal studies have revealed that OctoVAX microspheres are well-tolerated and induce no systemic toxicity effects.9 Furthermore, local reactogenicity was limited to minor foreign body reactions, as to be expected for any biodegradable implanted material. Therefore, it can be concluded that OctoVAX microspheres are well-tolerated and safe controlled-release vehicles that can be applied for the development of single-shot vaccines.
The development of a single-shot vaccine technology could contribute to a significant increase in vaccination coverage worldwide by improving patient compliance and lowering administration costs. To achieve this innovation, single-shot vaccines must be rationally designed. To facilitate this, complex factors in the development and manufacturing process that have a significant influence on the efficacy of the immune response must be controlled precisely during the development and manufacturing phases.
The concept of single-shot vaccination is applicable to various types of antigens and vaccines. Further research is required to investigate the influence of adjuvants and release characteristics in various vaccination regimens.
Bas Kremer, PhD, is a scientist in vaccine development, Rianne Roukema is a manager of corporate communications, and Leo de Leede is a director of preclinical research and development, all at OctoPlus NV, Leiden, the Netherlands, +31.71.5241071, email@example.com
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