3.5 Quality of vaccines
After cultivation, bacteria were harvested as previously described and
samples were analyzed according to standardized parameters (Gonçalves et
al., 2014). They all met the established Specification Criteria for
Acceptance (data not shown), including the parameter of the percentage
of soluble protein, which should be less than 15% (Lu et al., 2010a),
indicating that bacterial death was mediated by BPL rather than by
starvation. We also analyzed the protein profile of each lot produced
using different fermentation strategies by Western blot (Figure 5). A
panel of antibodies induced against potential vaccine candidates such as
PspA, PdT and PppA, or IL-17-inducing proteins, such as SP0785, SP2070,
SP2145 and SP1572, or even anti-PWCV sera were used. We observed that
all lots presented similar amounts of specific proteins, with the
expected size, independently of which process was used, and all were
comparable to our standard, the original vaccine lot produced in 60 L in
a batch process (Gonçalves et al., 2014).
In order to verify if different fermentation/downstream strategies would
produce effective vaccines, different preparations were evaluated for
the induction of IgG antibodies and IL-17A production in immunized
animals. Mice immunized with PWCV from lots prepared by any of the
fermentation strategies were equally immunogenic, producing high titers
of IgG and IL-17A (Figure 6A and B). In fact, PWCV obtained from the
perfusion-batch integrated to cell separation induced statistically
higher antibodies and IL-17A titers than the standard vaccine, the cGMP
lot produced at 60 L (Gonçalves et al., 2014) (Figure 6A and B, 2 and
6). This may be somehow due to a higher quality of the vaccine produced
in the integrated process, as 100 µg of total protein was given as
vaccine dose to all animals. One hypothesis is that the constant removal
of inhibitory metabolites throughout the integrated process led to lower
acetate and lactate concentration at the end of the culture, which would
be beneficial not only for the cell growth, but also for the production
of important antigens. On the other hand, metabolites accumulated in
batch and fed-batch processes, probably affecting antigen synthesis
besides inhibiting cell growth. Potency of these vaccines was evaluated
by challenge in the fatal aspiration model. All lots independently of
the fermentation strategy protected mice against challenge (Figure 6C),
indicating that the process intensification did not impact the quality
of vaccine.
The results of different downstream processes and storage temperatures
evaluation were also presented in Figure 6. Again, all lots were equally
immunogenic and induced high IgG and IL-17A titers (Figure 6D and E).
The lot obtained from the fed-batch process was the only one that
induced higher IL-17A titers than the positive control (cGMP lot
produced at 60 L) (Figure 6E, 2 and 7). Vaccine produced in batch
process with bacteria harvested by microfiltration induced statistically
higher IL-17A titers than the lot using the same fermentation strategy
and bacteria harvested by centrifugation (Figure 6E, 10 and 11). It is
worth noting that, after heating for BPL degradation, the final product
obtained using centrifugation for cell separation was not as homogenous
as before heating, whereas the same product obtained by microfiltration
was homogenous before and after heating for BPL degradation. When
centrifugation was applied, small particles or clumps were observed
after heating for BPL degradation. These clumps were not present during
the washing steps and after cell inactivation period, 30 h at 4 °C. We
conclude that this phenomenon was only observed when bacteria were
harvested by centrifugation, thus this methodology may compromise the
quality of the final product. Nevertheless, all lots protected
> 90% of immunized mice (Figure 6F).
4. Conclusion
In our studies, perfusion-batch with cell recycling integrated to the
cell separation process was the best promising strategy for the
production of PWCV, producing 3-fold higher biomass than batch and
fed-batch processes and 4-fold greater number of doses than the
previously described batch process, in which cells were harvested at OD
6.0. The perfusion-batch strategy supported the growth of S.
pneumoniae RM200 by removing inhibitory metabolites from the culture
and supplying nutrients. The integration of the perfusion with cell
separation system could be a cost-effective alternative to produce high
amounts of PWCV doses, using the same space and equipment as batch or
fed-batch cultures, also diminishing the auxiliary time. Therefore, the
process intensification achieved in this study has high potential for
scale up and bulk production of PWCV, as well as of other whole-cell
vaccines, which could be especially important to attend an
epidemiological emergency, when a high number of vaccine doses are
needed in a very short period of time. Moreover, the potential impact of
process intensification was carefully evaluated with respect to the
quality of the final product and no quality differences among the
vaccines were observed. Therefore, the perfusion-batch cultivation with
cell recycling integrated to the cell separation process should be
explored for large-scale production of PWCV for human immunization.