Manufacturing Process Development for an Epidermal Growth Factor-Based Cancer Vaccine - By incorporating disposable technologies and an improved purification scheme, scale-up and validation problems w

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Manufacturing Process Development for an Epidermal Growth Factor-Based Cancer Vaccine
By incorporating disposable technologies and an improved purification scheme, scale-up and validation problems were resolved.


BioPharm International Supplements


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. 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.
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 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).
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 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.


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