This purification step also made it possible to remove excess autologous protein (low molecular weight conjugates) from the
vaccine preparation by using higher cut-off membranes, with the aim of producing a more homogeneous vaccine drug substance.
The rationale behind this purification of the mixture of conjugation species was that the excess of autologous protein (rEGF),
either in form of polymers or as free molecules, does not contribute to the immuno-genicity of the vaccine preparation, and
instead dilutes the immunological action of the chemical conjugated rEGF-rP64k.
Figure 2 shows the anti-rEGF immunological response in mice immunized with a) the vaccine preparation for POC trials, b) the
vaccine preparation for the ASD process, or c) fractions of rEGF polymers and free rEGF molecules. Mice immunized with free
rEGF or rEGF polymers did not show anti-rEGF antibody responses, whereas mice immunized with both vaccine preparations did.
It was also observed that the vaccine preparation for the ASD process showed higher anti-rEGF antibody responses in mice when
compared with the vaccine preparation for POC trials. These results confirmed that it made sense to carry out ultrafiltration
of the conjugation mixture with higher cut-off membranes, to remove excess autologous protein from the vaccine preparation.
Figure 2. Anti-rEGF immunological response in mice immunized with a) the vaccine preparation for proof-of-concept (POC) trials;
b) the vaccine preparation from advanced stages of development (ASD); c) fractions of rEGF polymers and free rEGF molecules.
The UF–DF purification step thus provided a more homogeneous vaccine composition, rich in immunologically relevant species
(rEGF–rP64k). Figure 3 shows the molecular exclusion chromatographic profile of the products obtained by both processes. As
can be observed, the product made for POC trials yielded a chroma-tographic profile in which 42% of the total area belonged
to the rEGF-rP64k conjugated species (Figure 3A, peak 1). On the other hand, the vaccine preparation for the ASD process resulted
in a chromatographic profile in which the conjugated species (rEGF–rP64k) corresponds to 86% of the area (Figure 3B, peak
Figure 3. The molecular exclusion chromatographic profiles of the products obtained by the two different manufacturing processes.
The product prepared for proof-of-concept trials yielded a chromatographic profile in which 42% of the total area corresponded
to the rEGF-rP64k conjugated species (chromatogram A, peak 1). The vaccine preparation for advanced stages of development
resulted in a chromatographic profile in which the conjugated species rEGF-rP64k corresponded to 86% of the area (chromatogram
B, peak 1).
In addition, the UF–DF step is used to concentrate the protein mixture, allowing a wider range of potency and dose adjustments
for the vaccine preparation for different clinical settings.