Purification of polysaccharide conjugated vaccines using membrane filtration
Open Access
- Author:
- Emami, Parinaz
- Graduate Program:
- Chemical Engineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 09, 2020
- Committee Members:
- Andrew Zydney, Dissertation Advisor/Co-Advisor
Andrew Zydney, Committee Chair/Co-Chair
Ali Borhan, Committee Member
Themis Matsoukas, Committee Member
Craig Cameron, Outside Member
Seong Han Kim, Program Head/Chair - Keywords:
- Polysaccharide protein conjugate
Vaccine
Ultrafiltration
Diafiltration
Membrane fouling
Intermediate pore blockage
Microfiltration - Abstract:
- Polysaccharide-based (glycoconjugate) vaccines can provide effective immunization against diseases caused by a wide range of bacterial pathogens such as Streptococcus pneumoniae and Neisseria meningitidis. These pathogens are among the leading cause of childhood death in the world, with about one million deaths each year. There is a critical need for robust and cost-effective manufacturing processes to make these vaccines accessible for worldwide use. This thesis examined two of the critical steps in the manufacturing process for these vaccines: the separation of free residual polysaccharide from the glycoconjugate vaccine and sterile filtration of the purified drug substance using several glycoconjugates provided by Pfizer. Initial experimental studies were focused on understanding the factors governing the separation of the free polysaccharide from the glycoconjugate by ultrafiltration, including the effect of module geometry on the transmission of the free polysaccharide. Experiments were performed with several polysaccharides in both stirred cell and tangential flow filtration modules, with the latter being scaled down versions of ultrafiltration modules suitable for large-scale manufacturing. The transmission of a neutral serotype was unaffected by the module geometry except for the expected differences in the extent of concentration polarization. In contrast, the transmission of a highly charged polysaccharide was much lower in the tangential flow filtration systems due to membrane fouling associated with the flow induced elongation of the charged polysaccharide. Experimental studies were performed to identify appropriate conditions for the separation of the free and conjugated polysaccharide, including ionic strength, membrane pore size and chemistry, and feed and filtrate flow rates. Best performance was obtained at higher ionic strength and low feed flow rate to enhance the transmission of the free polysaccharide through the semipermeable membrane. A diafiltration process was developed that provided almost complete removal of the free polysaccharide with more than 70% yield of the glycoconjugate vaccine, with less than 25% of the diavolumes used in previous studies. Sterile filtration is used to ensure complete removal of bacteria from the purified glycoconjugate, but membrane fouling can cause low capacities and significant yield loss. The location of deposited glycoconjugate within different sterile filters was examined by confocal microscopy using fluorescently labeled versions of the glycoconjugates. Results with the homogeneous Durapore membrane showed capture of the glycoconjugates in a narrow band near the entrance of the membrane, with the rate of fouling determined by the specific properties of the glycoconjugate, most specifically the number of large particles in the feed solution. These results were used to develop a new fouling model based on a modification of the intermediate pore blockage framework. This model was in good agreement with the pressure profiles evaluated during constant flux filtration, providing a framework for the sizing and scale-up of sterile filtration processes for the production of glycoconjugate vaccines.