Fermentation Process
The final decision on the expression system of choice will be determined by the effect of post-translational modification
(PTM) on product efficacy. However, where there are no specific PTM requirements, microbial-based production platforms gain
an advantage. Previous resistance to using such systems, for instance in the production of Fab-based therapies have centred
around the limitation for manipulation of vH to vL expression levels, which can affect both productivity and product efficacy.
However, advances in transcriptional regulatory elements mean that coordinated control of these and other multicomponent products
can be optimized to significantly increase process titers while assisting in the decrease of normally associated product impurities.
In the VLP vaccine arena, various companies have focussed their attention on decreasing development timelines while increasing
product titers through the use of alternative cell-culture–based systems. Novavax, Inc. (Rockville, MD), has focussed on a
rapid development strategy for a VLP product targeted toward emerging influenza viruses, which has resulted in a concomitant
increase in both product potency and significant relative dose yields compared to the traditional egg-based manufacturing
systems. An effective vaccine can be manufactured in approximately 10–12 weeks from the point of strain identification while
clearly the requirement for scale in response to required doses has been significantly reduced. However, typical process (raw
material) cost of goods may still remain high.
More recently, the development of effective vaccine treatments through yeast and E. coli-based VLP production platforms have further decreased development and manufacturing timelines and scale requirements while
further increasing patient safety. With the development of platform downstream processing strategies and estimated recoveries
in the region of 50–60%, even small-scale bioreactors remain a firm target for manufacturing campaigns using these systems.
Indeed, estimated dose yields post purification are in the region of 500–1,000/L while development and manufacturing program
timelines are expected to halve those associated with historical tissue-culture–based systems.
Downstream Processing
Figure 3. Platform purification strategy for the production of virus like particles
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A platform approach to the purification process can also be adopted. The size of the particles can be advantageous during
the purification process allowing generic steps to be developed. One such purification strategy is outlined in Figure 3.
The process broadly consists of cell harvest to separate the cells from the fermentation media, resuspension into a controlled,
buffered environment, and cell lysis through high-pressure cell disruption. The bulk of the cellular debris is then cleared
from the solution by microfiltration and the process solution concentrated and buffer exchanged using a high molecular weight
cut-off TFF system. Further purification and polishing can then be achieved using anion exchange (AEX) and size exclusion
chromatography.
Harvest and Cell Disruption
VLPs are produced as an intracellular product from a microbial production system. They must be released into the process stream
to allow for subsequent purification. This can be achieved by several means, the most common being the use of high-pressure
lysis. This is a process whereby the process solution is forced through a small fixed orifice at high pressure. The rapid
transfer of the sample from a region of high pressure to one of low pressure causes cell disruption.
This type of cell disruption is by far the most efficient and reproducible but does have a number of drawbacks in processing
of VLPs. The extremely high shear not only lyses the cells but also micronizes the cellular debris; this coupled with the
large size of the VLPs themselves can make the subsequent clarification step problematic. Careful attention must be paid during
the development of such a step to strike a balance between cellular disruption and the release of the VLPs into the process
medium and the micronization of cellular debris to avoid fouling issues further downstream in the production process.
Clarification
A number of options are available for clarification including centrifugation, depth filtration, or micro-filtration to name
a few. Generally, microfiltration is the preferred option as it is well established in the biopharmaceutical industry as a
scalable and robust clarification technique. Whichever technique is used, this clarification step may be problematic post
high-pressure lysis.
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