ULTRAFILTRATION OF BACTERIAL POLYSACCHARIDES AND PROTEIN-POLYSACCHARIDE CONJUGATES USED IN VACCINES
Open Access
- Author:
- Hadidi, Mahsa
- Graduate Program:
- Chemical Engineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- July 27, 2016
- Committee Members:
- Andrew Zydney, Dissertation Advisor/Co-Advisor
Andrew Zydney, Committee Chair/Co-Chair
Themis Matsoukas, Committee Member
Ali Borhan, Committee Member
Craig Eugene Cameron, Outside Member - Keywords:
- Polysaccharide-protein conjugate
Vaccine
Ultrafiltration
Size Exclusion Chromatography
Capsular polysaccharide
Pneumococcus
Fouling
Ionic strength
electrostatic interactions - Abstract:
- Polysaccharide-based vaccines can protect against various diseases such as pneumonia and meningitis. In order to increase the effectiveness of the vaccine, bacterial capsular polysaccharides are covalently attached to an immunogenic protein, resulting in a protein-polysaccharide conjugate vaccine. Purification of these very large biomolecules can be a significant challenge in commercialization of these vaccines. The overall objective of this dissertation was to develop a fundamental understanding of the potential of using membrane ultrafiltration for the separation / purification of these polysaccharides and their corresponding conjugates. Experiments were performed using several pneumococcus polysaccharide serotypes provided by Pfizer. Ultrafiltration data were obtained in a stirred cell using cellulose and polyethersulfone membranes with different molecular weight cutoffs. Polysaccharides were characterized using dynamic light scattering and size exclusion chromatography. Polysaccharide transmission in dilute solutions was a strong function of filtrate flux due to concentration polarization effects, with the data in good agreement with available hydrodynamic models. Polysaccharide fouling became significant at high filtrate flux using more concentrated solutions, consistent with the presence of a critical wall concentration for fouling that was dependent on the specific serotype. The flux and polysaccharide transmission were both strong functions of solution ionic strength due to a combination of inter- and intra-molecular electrostatic interactions. The polysaccharide sieving coefficients were well-correlated with the effective polysaccharide size as determined by size exclusion chromatography, with results in good agreement with available hydrodynamic models for membrane transport. The increase in effective size at low salt concentrations could be explained using the worm-like chain model accounting for the increase in persistence length and excluded volume at low ionic strength. These results provide important insights into the factors controlling the ultrafiltration behavior of bacterial polysaccharides as well as an initial framework for the design of membrane processes for purification of polysaccharide-based vaccines.