Sterile filtration process for glycoconjugate vaccine drug substances and drug product
Open Access
- Author:
- Du, Zhuoshi
- Graduate Program:
- Chemical Engineering (PHD)
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- January 23, 2024
- Committee Members:
- Robert Rioux, Professor in Charge/Director of Graduate Studies
Andrew Zydney, Chair & Dissertation Advisor
Esther Gomez, Major Field Member
Yuguo Lei, Outside Unit & Field Member
Stephanie Velegol, Major Field Member - Keywords:
- Glycoconjugate Vaccine
Drug Substances
Drug Product
Sterile Filtration
Membrane Fouling
Prefiltration
Scale-up
Sterile Filters Screening - Abstract:
- Glycoconjugate vaccines containing multiple serotypes of a bacterial capsular polysaccharide can provide strong immune protection against pathogenic infections. Sterile filtration is an important component of the fill and finish operations in the preparation of these vaccines, ensuring that the final drug product contains no pathogenic bacteria. The capacity of the sterile filter is limited by membrane fouling, but there is currently little understanding of the factors controlling the fouling behavior during sterile filtration of these important multivalent glycoconjugate vaccines. The overall objectives for this dissertation include: (1) evaluate the performance of the sterile filtration process using both the individual drug substances and the multivalent drug product, (2) investigate the fouling mechanisms and develop models for prediction of the filtrate flux / transmembrane pressure during the glycoconjugate vaccine sterile filtration process, (3) develop strategies to increase the capacity of the sterile filtration step, (4) compare the fouling behavior of different commercial sterile filters, and (5) examine issues involved in the scale-up of the sterile filtration process for glycoconjugate vaccine products. Experimental studies were performed to explore the potential for significantly increasing the capacity of the sterile filter through the use of an appropriate prefilter. Data were obtained using a single serotype drug substance with prefilters having different pore size and chemistry, with the sterile filtration performed at constant filtrate flux using 0.22 µm nominal pore size Durapore® polyvinylidene difluoride membranes. Prefiltration through 5 µm pore size Durapore® or Nylon prefilters nearly eliminated the fouling of the sterile filter, leading to more than a 100-fold reduction in the rate of pressure increase for the sterile filter. This dramatic improvement in sterile filter performance was due to the removal of large components (greater than 1 µm in size) as confirmed by dynamic light scattering. The fouling behavior of the multivalent drug product, which contains 4 different glycoconjugate serotypes, was examined by confocal microscopy by appropriately labeling the individual serotypes with different fluorescent dyes. The resulting confocal images demonstrate that all four serotypes deposit in a narrow band near the inlet of the Durapore® filter. The much lower conductivity of the formulation buffer (compared to the buffers used with the individual drug substance) had a large effect on the fouling behavior for some, but not all, of the serotypes. In addition, the fouling resistance associated with the drug product was greater than the sum of the resistances of the individual serotypes reflecting the simultaneous fouling (pore blockage and cake filtration) by the 4 serotypes in the multivalent glycoconjugate vaccine product. The filtration behavior of a range of commercial 0.2 / 0.22 µm nominal pore size sterilizing grade filters with both single-layer and dual-layer structures were examined using the multivalent glycoconjugate vaccine drug product. The highly asymmetric Millipore Express® showed much greater capacity than the more homogeneous filters, with the support structure of the Express® acting as a prefilter that is able to remove foulants thereby protecting the small pores in the size-selective skin layer. This behavior was confirmed by performing experiments with different batch prefilters and by examining the location of foulant deposition within the different sterile filters using confocal microscopy. Scale-up experiments were conducted during constant flux filtration through 0.22 µm Durapore® polyvinylidene difluoride (PVDF) membranes, both with small discs and with the Opticap® XL2 pleated cartridge. The transmembrane pressure increased rapidly during the glycoconjugate filtration due to membrane fouling, with the rate of pressure increase being more pronounced in the pleated cartridge. Additional insights into the fouling behavior were obtained using confocal microscopy by in situ labeling of the glycoconjugate captured within the filter media using an Alexa Fluor fluorescent dye. Glycoconjugate deposition occurred only within the first 5–15 µm of the 0.22 µm Durapore® membrane at both scales, with more variability in the deposition pattern observed for the pleated filter due to the non-uniform flow distribution in the Opticap® XL2 cartridge. These results provide important insights into the underlying fouling behavior during sterile filtration of glycoconjugate vaccines as well as a framework for the design, optimization, and scale-up of the sterile filter step in glycoconjugate biomanufacturing.