Monitoring of an Antigen Manufacturing Process Using Fluorescence

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