Integration of PAT in Biopharmaceutical Research: A Case Study - Pharmaceutical Technology

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Integration of PAT in Biopharmaceutical Research: A Case Study
This case study describes the implementation of process analytical technology on the cultivation process step of a whole-cell vaccine against whooping cough disease.

Pharmaceutical Technology
Volume 33, Issue 7

Evaluating product quality

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.
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.

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).


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