Formulation and Manufacturing of Single-Shot Vaccines
Figure 3. Schematic representation of the microsphere preparation process
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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 4. Bioreactor used during scale-up of the production process
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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.
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
Controlling Particle Size During Process Scale-up
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.
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 μm.
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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.
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.
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