On-line monitoring of product quality and continuous process optimization strategies are common in many industries, but only recently has the US Food and Drug Administration recognized the need for the pharmaceutical industry to come to the same level. To meet this need, FDA launched the process analytical technology (PAT) guideline (1), which prescribes quality assurance to become part of the manufacturing process, referred to as quality by design (QbD), in contrast to quality by testing, which relies on the postproduction testing and release of the product. Under QbD, the observed processes need to be well-characterzed, and manufacturers need to know the critical attributes of each process, each product, and their interactions (2).
The European Medicines Agency (EMEA) and the Japanese regulatory authorities have adopted PAT principles, which, through the International Conference for Harmonization (ICH), resulted in three new guidance documents (Q8, Q9, and Q10). These documents are now considered the world standard for this new regulatory concept (i.e., QbD with PAT) for product and process quality (3). For pharmaceutical small molecules with relatively simple production processes, the application of PAT is becoming increasingly common. Acceptance of PAT concepts has been much slower for biological products. FDA has therefore invited biopharmaceutical companies to work with them for pilot submissions of biopharmaceutical PAT applications (4).
The Netherlands Vaccine Institute's (NVI) PaRel project is a good example of a successful PAT implementation in a bioprocessing environment. The project's aim was to implement PAT in the cultivation process step in making a whole-cell vaccine used to prevent whooping cough. This process step involves batch cultivation of the Bordetella pertussis bacterium—the most complicated and critical step in this production process and, as such, was chosen for the development of the tools, equipment, and knowledge necessary for full PAT application. Compared with a small-molecule drug, this vaccine is relatively undefined and complex, which makes it more difficult to ensure product quality during processing. FDA has acknowledged that the approach chosen in the PaRel project can lead to approval of a PAT application for a complex biopharmaceutical product such as a whole-cell vaccine.Integrating PAT into cell cultivation
Understanding the cell-cultivation process requires an understanding of the interior physiology of the cell and how it responds to external changes, either because of process changes or disturbances. This understanding is important, especially in a batch-wise operated bioreactor, where continuously changing conditions may influence cellular physiology. To achieve this, a full genome DNA microarray analysis was developed for monitoring messenger RNA (mRNA) expression profiles of B. pertussis. By measuring gene expression levels during cultivation, the pathways or specific proteins affected by changes during processing can be identified. In this way, disturbances of specific process parameters can be assessed for impact on product quality. It is also possible to make determinations regarding reproducibility between batches and the optimal harvest point by examining the gene expression profile.
In a whole-cell vaccine, the outer membrane proteins are the targets presented to the immune system; therefore, the outer membrane protein composition is crucial for vaccine quality. Streefland et al. described a method for identifying genes that encode those outer membrane components known to induce a protective immune response (5). A highly conserved molecular switch, called the Bordetella virulence gene (Bvg) system, regulates the virulence genes of the Bordetella genus. This means that a single extracellular signal can induce the complete physiological switch between the virulent and the nonvirulent state (6). All genes involved are controlled by the same operon system. For some strains, these virulence genes have already been investigated (6, 7), but not in terms of optimal outer membrane composition for vaccine manufacturing.
The dissolved oxygen concentration was one of the process parameters investigated in the PaRel project. Because B. pertussis is an obligate aerobic organism, the availability of oxygen during cultivation is essential. To investigate this, a series of cultivations were oxygen-limited for 90 min, and samples were taken immediately before and after limitation as well as at the end of cultivation for microarray analysis. Oxygen limitation had a strong effect on gene expression levels of many genes immediately after the event, including virulence genes. At the end of cultivation, however, the oxygen-limited cultures could not be distinguished from the standard control cultivations. This indicates that oxygen limitation can have an effect on vaccine quality, but this effect is reversible. As long as oxygen limitation does not occur at or near the end of cultivation, it is not a crucial factor in manufacturing the vaccine (8).
Using microarrays to monitor crucial genes enabled us to analyze the critical process attributes for the cultivation of B. pertussis. High expression of the 56 virulence marker genes is associated with high cellular virulence and thus good expected product quality. Low expression levels are associated with poor expected quality. Therefore, a weighted average of the expression levels of these virulence marker genes can be used to predict the quality of the bacterial suspension at the end of cultivation. This so-called product quality score can be used to compare objectively batches or samples of the same batch with each other.