Clinical experiences with CIMAvax-EGF in advanced lung cancer patients demonstrated that vaccination provoked an increase
in anti-EGF antibody titers and a decrease in EGF sera concentration. The increase in anti-EGF antibodies directly correlated
with increased survival of vaccinated patients. Decreases in EGF sera concentration also correlated with increased survival
of vaccinated patients.14,17, 19 In randomized controlled trials, it was demonstrated that overall, more vaccinated patients survived than non-vaccinated
controls, an effect that is much greater in patients under 60 years old.19
The long path for translating this basic concept into a real product (CIMAvax-EGF) began in 1992. The first challenge was
trying to prepare an immunogenic preparation with a self antigen, because any cancer vaccine based on self antigens must be
designed to create an adequate presentation environment to provoke a clinically significant immune response.9
To achieve this, the self protein (EGF) was conjugated to a immunogenic carrier protein. Several carrier proteins were tested
in the preclinical10,11 and clinical settings. 12–14,17–19 Based on immunogenicity results, the recombinant protein rP64k from Neisseria meningitides was selected for continued product development.13
Two adjuvants, aluminum hydroxide and Montanide ISA-51 (Seppic, France), were tested. The best results, in terms of immunogenicity
of the vaccine formulation, were obtained using Montanide ISA-51, so this adjuvant was selected for further product development.13
For development of this vaccine through proof-of-concept (POC) clinical trials, a fairly simple manufacturing process was
used. This initial production process consisted of chemical conjugation using a linker reagent, followed by an impurity-removal
step with a dialysis membrane. However, this process had practical disadvantages for scale-up and compliance with good manufacturing
practice (GMP) requirements.
As the product development cycle advanced to late clinical trials, the manufacturing process needed to be improved to comply
with GMP requirements and undergo validation. The challenge was to develop a new process for advanced stages of development
(ASD) while maintaining the performance equivalence with the vaccine preparation used for the POC studies.
The process development strategy focused on the following goals:
- Optimizing the conjugation reaction
- Replacing the membrane dialysis purification process with a step that could be scaled up more easily
- Incorporating disposable tech-nology to further facilitate scale-up and cleaning validation
- Improving process and product characterization
- Evaluating the process con-sistency of the new process
- Evaluating the equivalence of the vaccine preparations.
Optimizing the Conjugation Reaction
The chemical conjugation method developed for the POC vaccine preparation allowed unspecific binding of the immunogenic carrier
protein (rP64K) and the autologous protein (rEGF). This procedure required a high molar quantity of rEGF to avoid reaching
the reaction limit. To ensure that the conjugation reaction yielded reproducible conjugation products following scale-up,
conjugation reaction kinetics were studied to understand reaction times, the effect of reactant feeding strategies, and mixing
requirements. As a result of the optimization studies, mixing and reaction times were defined that allowed reproducible conjugation
results following scale-up of the process more than ten- fold. The working ranges for the main process variables were set
for commercial operation following robustness studies.
Replacing the Membrane Dialysis Purification Step
Most of the limitations of the process used to manufacture product for the POC studies resulted from the membrane-based dialysis
purification step, which was designed to remove the free conjugation reagent and other chemical substances before final formulation.
The main drawbacks of this step were extended process time, limited scalability, and the inclusion of several manual operations
in the process.
To overcome these difficulties, a purification step based on crossflow membrane ultrafiltration–diafiltration (UF–DF) was
introduced as an alternative to the dialysis membrane.
This UF-DF procedure reduced processing time and significantly reduced the risk of microbial contamination. It also made it
possible to perform clean-in-place operations. Furthermore, this purification alternative could be scaled up in a linear fashion
to adapt to varying batch volumes.