Introducing Disposable Technology into the Process
Disposable technology was introduced into the process to provide flexibility for scale-up and facilitate GMP compliance. Disposable
filters and bioprocess containers (bags) were introduced into the manufacturing process to support operations such as mixing,
buffer preparation, and buffer and bulk storage.
The steps of final sterile filtration and aseptic filling also were improved by using disposable technology. A list of single-use
products incorporated into various steps in the vaccine manufacturing process is shown in Table 1.
Table 1. Disposable products introduced into the manufacturing process
Improved Process and Product Characterization
During translation of the POC vaccine formulation into the ASD product, a set of analytical tests was developed for product
characterization and for quality control release of the purified bulk and final product (Figure 4). For antigenic identity,
the presence of conjugated species (rEGF–rP64k) was detected by SDS/Western blot. For this purpose, we developed a specific
Figure 4. Process flow chart for the CIMAvax-EGF manufacturing process for advanced stages of product development. The in-process
controls and the quality control test for final product and biological starting materials (rEGF and rP64k proteins) are included
in the corresponding steps.
The content of immunogenic species (rEGF–rP64k) was determined by HPLC–GF. Quantification of total protein concentration was
performed using the Lowry method. To evaluate residual glutaraldehyde content, an HPLC–RP assay was developed using a C-8
Immunogenic activity tests are a significant challenge in human cancer vaccines based on a self protein. The difficulty is
greater for in vivo tests, because what is self for humans is not self for other animal species. In our case, we had the advantage that the outbred
mice strain (NMRI) only developed anti-rEGF responses when immunized with the conjugated rEGF–rP64k, and did not do so when
immunized with rEGF alone. We then developed a test by immunizing NMRI mice, in which immune responses demonstrated the immunogenicity
not only of the antigen, but also of the conjugation of both species (rEGF and rP64k) .
Evaluating Process Consistency
The analytical data (Table 2) showed that the process was able to produce consecutive vaccine lots with consistent performance.
They also showed the equivalence between the products manufactured for POC trials and ASD.
Table 2. Comparative analytical results of the final product tests for the two vaccine preparations