Bordetella pertussis is one of two Gram-negative bacteria responsible for causing whooping cough in humans, a highly contagious disease that infects the human upper respiratory tract. Whole-cell and acellular vaccines have been developed but due to side-effects resulting from whole-cell vaccines, acellular vaccines are currently preferred to prevent this disease. A second bacterium known to cause whooping cough is Bordetella parapertussis, but since it causes less aggressive symptoms, only B. pertussis is utilized in the manufacture of the vaccine. One acellular vaccine is based on four virulence factors: pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), and fimbriae (FIM). The focus of this thesis was to explore the use of spectrofluorometry for monitoring and forecasting the performance of the upstream and downstream operations in the PRN purification process at Sanofi Pasteur. The upstream fermentation process includes a series of reactors of increasing volume where the microorganism is grown under controlled conditions. The PRN purification process involves a series of sequential steps for separating this protein from other proteins for later use in the vaccine. The PRN is precipitated in three steps with ammonium sulphate at three different concentrations. The pellet is collected by centrifugation and dissolved in a buffer solution followed by chromatographic separation. The run-through is then ultra-filtered and diafiltered in two separate steps. The resulting concentrate is dissolved in water and subjected to another chromatographic step and diafiltration. The final filtration of PRN involves a pre-filtration and sterile filtration. Finally, the samples are collected for quality control. The objective of this work was to monitor the process at different steps of the upstream and downstream purification process by multi-wavelength fluorescence spectroscopy in combination with multi-variate statistical methods. From the spectra, it was possible to identify fluorescent compounds, such as amino acids and enzyme cofactors, without performing an additional pre-treatment or purification step. Also, the identification of conformational changes in proteins and formation of complexes, such as NAD(P)-enzyme complex, was possible based on the shift in the emission peaks of the compounds identified. These results demonstrated the feasibility of using this tool for qualitative evaluation of the process. Multivariate methods, such as PCA and PLS, were used to extract relevant information and compress the fluorescence data acquired. PCA was effective for correlating variability in the yield of pertactin to a particular fluorescence fingerprint. As a result of the study, it was concluded that a possible source of variability in the productivity that is observed might be a metabolic shift during the fermentation steps that leads to the accumulation of NAD(P)H (or NAD(P)H-enzyme complex) probably due to oxygen transfer limitations. This conclusion was reached after investigating changes in the dissolved oxygen, aeration, agitation, supplementation time and key metabolites (lactate, glucose, glutamine) profiles. The correlation of these parameters with low productivity it was not straightforward; however, some consistencies were observed, for example, high levels of glutamine in batches with low productivity. This fact might be related to the start of the supplementation time, which may be related to the dissolved oxygen, since the addition of the supplement is done manually when an increase of the dissolved oxygen is detected. It is believed that this factor is related to the low production of protein product, such as pertactin. By means of PLS, it was possible to build regression models that allow for predicting the final concentration of pertactin from the fluorescence measurements. The models were built using the new variables obtained from data compression performed with PCA, and the final pertactin concentration measured by a Kjeldahl test. With this method, two regressions were constructed: (i) between NAD(P)H-enzyme complex spectra from the fermenters and pertactin concentration and (ii) between the pertactin fluorescence spectra from the last step of purification and pertactin concentration. A third model was built using the protein content, the NAD(P)H-enzyme complex content in the fermenters and pertactin concentration. Attempts were made to identify the possible enzyme that may bind to NAD(P)H, assumed to be a dehydrogenase. Substrates for different enzymes were employed with the objective of measuring changes in the fluorescence of the characteristic peak for this binding (Ex/Em=280/460 nm). Major changes were detected after addition of the substrates oxaloacetate, ubiquinone and succinate dehydrogenase. Since changes were detected with more than one substrate, it was not possible to unequivocally identify the enzyme; however, the results provide some insight into what may be happening at the metabolic level. The work carried out in this thesis involved both analysis of samples provided or collected by the industrial sponsor as well as analysis of samples prepared at the University of Waterloo for measurement, interpretation and calibration. The proposed fluorescence-based method was found suitable for assessing protein quantity as well as for providing an indication of possible protein aggregation and conformational changes. Future work will be required to identify the exact source of variability in the production of pertactin, by means of monitoring the evolution of fermentation, NAD(P)H and ATP measurements, and oxidation redox potential assays.

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During the past two decades, many researchers have developed methods for the detection of structural defects at the early stages to operate the aerospace vehicles safely and to reduce the operating costs. The Surface Response to Excitation (SuRE) method is one of these approaches developed at FIU to reduce the cost and size of the equipment. The SuRE method excites the surface at a series of frequencies and monitors the propagation characteristics of the generated waves. The amplitude of the waves reaching to any point on the surface varies with frequency; however, it remains consistent as long as the integrity and strain distribution on the part is consistent. These spectral characteristics change when cracks develop or the strain distribution changes. The SHM methods may be used for many applications, from the detection of loose screws to the monitoring of manufacturing operations. A scanning laser vibrometer was used in this study to investigate the characteristics of the spectral changes at different points on the parts. The study started with detecting a load on a plate and estimating its location. The modifications on the part with manufacturing operations were detected and the Part-Based Manufacturing Process Performance Monitoring (PbPPM) method was developed. Hardware was prepared to demonstrate the feasibility of the proposed methods in real time. Using low-cost piezoelectric elements and the non-contact scanning laser vibrometer successfully, the data was collected for the SuRE and PbPPM methods. Locational force, loose bolts and material loss could be easily detected by comparing the spectral characteristics of the arriving waves. On-line methods used fast computational methods for estimating the spectrum and detecting the changing operational conditions from sum of the squares of the variations. Neural networks classified the spectrums when the desktop – DSP combination was used. The results demonstrated the feasibility of the SuRE and PbPPM methods.

During the past two decades, many researchers have developed methods for the detection of structural defects at the early stages to operate the aerospace vehicles safely and to reduce the operating costs. The Surface Response to Excitation (SuRE) method is one of these approaches developed at FIU to reduce the cost and size of the equipment. The SuRE method excites the surface at a series of frequencies and monitors the propagation characteristics of the generated waves. The amplitude of the waves reaching to any point on the surface varies with frequency; however, it remains consistent as long as the integrity and strain distribution on the part is consistent. These spectral characteristics change when cracks develop or the strain distribution changes. The SHM methods may be used for many applications, from the detection of loose screws to the monitoring of manufacturing operations. A scanning laser vibrometer was used in this study to investigate the characteristics of the spectral changes at different points on the parts. The study started with detecting a load on a plate and estimating its location. The modifications on the part with manufacturing operations were detected and the Part-Based Manufacturing Process Performance Monitoring (PbPPM) method was developed. Hardware was prepared to demonstrate the feasibility of the proposed methods in real time. Using low-cost piezoelectric elements and the non-contact scanning laser vibrometer successfully, the data was collected for the SuRE and PbPPM methods. Locational force, loose bolts and material loss could be easily detected by comparing the spectral characteristics of the arriving waves. On-line methods used fast computational methods for estimating the spectrum and detecting the changing operational conditions from sum of the squares of the variations. Neural networks classified the spectrums when the desktop – DSP combination was used. The results demonstrated the feasibility of the SuRE and PbPPM methods.

Les défauts récurrents apparaissant dans des contextes particuliers ont un impact non négligeable sur le rendement, lors de la fabrication des noeuds technologiques nanométriques. C'est pourquoi une nouvelle méthode de contrôle du procédé de fabrication in-situ est développée, en vue d'améliorer la performance globale de l'outil de production. Elle se veut complémentaire des techniques d'analyses de défaillance classique, notamment en cas de crise. Le principe est de transformer un circuit de production en un véhicule de test en réutilisant des éléments qui le composent. Le circuit perd alors sa fonctionnalité originale au profit de fonctions de contrôle du procédé de fabrication réalisées uniquement à base de cellules logiques disponibles en grand nombre dans les circuits. Cette transformation, baptisée «échange topologique», implique la modification de certains niveaux de métallisation et nécessite la création d'un flot de conception particulier, basé sur des techniques d'Engineering Change Order (ECO). Comme plusieurs fonctions doivent pouvoir cohabiter ensemble sur un même véhicule, un système de multiplexage a été évalué. La faisabilité est montrée au travers d'un circuit de test réalisé de manière analogue à un circuit en production. Il est transformé pour l'exemple en un système intégré de contrôle d'épaisseur de diélectrique. / Recurrent defects appearing in specific contexts have a significant impact on nanoscale technology nodes manufacturing process yield. Therefore, a new in-situ process monitoring method is developed to improve the overall performance of the production tool. It is complementary to classical failure analysis techniques, especially when a yield crisis occurs. The idea is to transform a production circuit into a test vehicle by reusing its components. The circuit loses its original functionality in favor of process monitoring functions, carried out only with standard cells widely available in circuits. This transformation, called "topological exchange" involves modifying some levels of metallization and requires the creation of a particular design flow, based on Engineering Change Order (ECO) techniques. As several functions must be able to cohabit on the same vehicle, a multiplexing system is evaluated. Feasibility is shown through a test circuit designed analogously to a production circuit. It is transformed for the example into an integrated dielectric thickness control system.