Evaluating product quality
The product quality score was used to determine a critical event in batch cultivation: the optimal harvest point (9). As biomass
accumulates exponentially, nutrient concentrations drop exponentially (see Figure 1), which can result in a range of unwanted
effects, including cell lysis or the suppression of virulence genes (10). To investigate the optimal harvest point, four identically
operated cultivations were each sampled at 11 time points for microarray analysis. A continuously changing gene expression
pattern was expected because of the continuously changing extracellular environment associated with batch cultivation. However,
gene expression proved to be relatively constant, which resulted in high product quality scores during most of the cultivation.
Toward the end of cultivation, the product quality score dropped sharply (see Figure 1). This drop coincided with the depletion
of the nutrients lactate and glutamate, a determination that allowed the optimal harvest point to be accurately predicted.
Therefore, measuring lactate and glutamate concentrations during cultivation demonstrated that the bacteria can be consistently
harvested before nutrients become limiting, assuring the optimal composition of the bacterial outer membrane.
Figure 1: Batch profile analysis for the cultivation of Bordetella pertussis. Diagram (a) shows the increase in cell numbers
for four cultivations and the decrease in average concentrations of the nutrients lactate and glutamate. Lines A to K in Diagram
A indicate the points at which samples for microarray analysis were taken. Diagram (b) shows the relative product quality
scores at the sample points A to K based on Diagram (a). It is clearly shown that the product quality score is consistently
high for most of the process. Toward the end of the cultivation (as shown by points J and K), the product quality drops sharply.
This decline corresponds with the depletion of important nutrients (Diagram a) in real time.
Critical process parameters
The definition of PAT states that it is a "system for designing, analyzing and controlling manufacturing through timely measurements
of critical quality and performance attributes of raw and in-process materials and processes, with the goal of ensuring final
product quality" (1). With the on-line measurement of nutrient concentrations and the supporting evidence that this correlates
with virulence gene expression and subsequently outer membrane composition, the requirements for a PAT application are fulfilled,
at least for the harvest-point determination. For other critical process attributes, several sensors are available to measure
pH or dissolved oxygen, for example. However, these sensors only monitor a single parameter, which means one could risk missing
a critical parameter or be required to mount numerous probes on the bioreactor system for each measured parameter. To monitor
many parameters simultaneously, a near infrared (NIR) spectroscopic probe that measured a spectrum between 800 and 2500 nm
was placed inside the bioreactor. The instrument's range, situated between visible light and far infrared, is sensitive for
chemical changes (band vibration overtones) and physical changes (combination bands) such as optical density, viscosity, particle
size, and particle morphology. This range makes the technique highly suitable for monitoring bacterial cultivation processes,
as was confirmed by initial pilot studies (11).