Scale-up issues during sterile filtration of glycoconjugate vaccines.

Several recent studies have provided important insights into the factors controlling the sterile filtration of glycoconjugate vaccines; however, this work has been limited to small-scale disk filters with very uniform flow distribution. The objective of this study was to examine the scale-up of the sterile filtration step using a glycoconjugate drug substance made from a single polysaccharide serotype. Experimental data were obtained 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 scale-up of the sterile filter step in glycoconjugate biomanufacturing.

Evaluation of single-use tangential flow filtration technology for purification of activated polysaccharides used in conjugate vaccine manufacturing.

Over the past decade, single-use tangential flow filtration (TFF) technologies have emerged to reduce system preparation time, promote fast and flexible product change over, and ultimately shorten process development and manufacturing time/cost. In this study, the performance of a recently developed Pellicon® single-use TFF capsule was compared against traditional Pellicon® cassettes by assessing TFF process performance (such as flux, residuals clearance, and yield) and post-purification product attributes (such as concentration and mass-weighted average molecular weight). Good scaling was shown by comparing process performance and product attributes across different scales and formats. Additionally, similar TFF process performance and post-purification product attributes were observed for the single-use capsule compared to the reusable TFF cassettes. The capsule requires a smaller flush than the cassette, and it is easier to use since it does not require a compression holder or pre-sanitization. The results provide insight into the application of the single-use TFF capsule and scalability of TFF processes for the purification of conjugate vaccines.

Prefiltration enhances performance of sterile filtration for glycoconjugate vaccines.

Recent studies have reported very low capacity during sterile filtration of glycoconjugate vaccines due to rapid fouling of the sterile filter. The objective of this study was to explore the potential for significantly increasing the capacity of the sterile filter through the use of an appropriate prefilter. Data were obtained using prefilters with 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. These results demonstrate the potential of using large pore size prefilters to significantly enhance the performance of the sterile filtration process for the production of important glycoconjugate vaccines.

Fouling Behavior during Sterile Filtration of Different Glycoconjugate Serotypes Used in Conjugate Vaccines.

PURPOSE: Sterile filtration can be a particular challenge when processing very large glycoconjugate vaccines. The objective of this study was to examine the sterile filtration performance of a series of glycoconjugate vaccines produced by coupling different polysaccharide serotypes to an immunogenic protein. METHODS: Sterile filtration was performed at constant filtrate flux using 0.22 µm pore size Durapore® polyvinylidene fluoride membranes. Glycoconjugates were characterized by dynamic light scattering, rheological measurements, and nanoparticle tracking analysis (NTA). Confocal microscopy was used to examine glycoconjugate capture profiles within the membrane. Transmembrane pressure data were analyzed using a recently developed fouling model. RESULTS: All glycoconjugates deposited in a narrow band near the entrance of the Durapore® membranes. The rate of fouling varied significantly for the different serotypes, with the fouling parameter correlated with the fraction of glycoconjugates larger than 200 nm in size. CONCLUSIONS: The fouling behavior and sterile filter capacity of the different glycoconjugate serotypes are determined primarily by the presence of large species (>200 nm in size) as determined by nanoparticle tracking analysis. The modified intermediate pore blockage model provides a framework for predicting the sterile filtration performance for these glycoconjugate vaccines.

Purification of a conjugated polysaccharide vaccine using tangential flow diafiltration.

Conjugated vaccines prepared from the capsular polysaccharide of Streptococcus pneumoniae can provide immunization against invasive pneumococcal disease, meningitis, and otitis media. One of the critical steps in the production of these vaccines is the removal of free (unreacted) polysaccharides from the protein-polysaccharide conjugate. Experimental studies were performed to evaluate the effects of membrane pore size, filtrate flux, and solution conditions on the transmission of both the conjugate and free polysaccharide through different ultrafiltration membranes. Conjugate purification was done using diafiltration performed in a linearly-scalable tangential flow filtration cassette. More than 98% of the free polysaccharide was removed within a 5-diavolume diafiltration process, which is a significant improvement over previously reported results for purification of similar conjugated vaccines. These results clearly demonstrate the opportunities for using ultrafiltration/diafiltration for the final purification of conjugated vaccine products.

Effects of Plasma Proteins on the Transport and Surface Characteristics of Polysulfone/Polyethersulfone and Asymmetric Cellulose Triacetate High Flux Dialyzers.

Blood-membrane interactions can have a large impact on the performance of hemodialysis membranes, particularly for high flux membranes in which the membrane itself provides very low resistance to solute transport. The objective of this study was to examine the effects of exposure to serum on the solute clearance and convective sieving characteristics of high flux polysulfone (Optiflux F250NR), polyethersulfone (ELISIO-25H), and asymmetric cellulose triacetate (SOLACEA-25H) hemodialyzers using both vitamin B12 and a range of polydisperse dextrans. Zeta potential measurements were used to obtain additional insights into the changes in membrane surface properties. Exposure to serum in a simulated dialysis session caused a significant reduction in both solute clearance and sieving coefficients for the polysulfone/polyethersulfone dialyzers, particularly for the larger molecular weight solutes. In contrast, the transport characteristics of the asymmetric cellulose triacetate dialyzers were almost unchanged after exposure to serum. The zeta potential of the cellulose triacetate membrane became slightly more negative after exposure to serum, consistent with an adsorbed protein layer composed largely of albumin. The net result is that the asymmetric cellulose triacetate dialyzer had dramatically higher clearance of the larger dextrans after exposure to serum, with the clearance and sieving coefficient for a 10 kDa molecular weight dextran being more than an order of magnitude greater than that of the polysulfone/polyethersulfone membranes. These results provide important insights into the expected clinical performance of these high flux dialyzers.

Transport Characteristics of Asymmetric Cellulose Triacetate Hemodialysis Membranes.

AIMS: The objective of this study was to compare the transport characteristics of highly asymmetric cellulose triacetate (ATA™) membranes with that of both symmetric cellulose triacetate and asymmetric polysulfone membranes. METHODS: Data were obtained for solute clearance and sieving coefficients of vitamin B12 and a range of polydisperse dextrans using ATA™ SOLACEA-25H and Optiflux F250NR polysulfone dialyzers. Results for these, and the CT190 symmetric cellulose triacetate dialyzer, were analyzed using available membrane transport models. RESULTS: The ATA™ had the largest solute clearance, although the homogeneous CT190 dialyzer had the highest sieving coefficients. These differences were a direct result of the differences in the underlying membrane morphology, with the asymmetric ATA™ membrane providing much higher diffusive transport rates (and thus higher solute clearance). CONCLUSIONS: These results demonstrate the importance of membrane morphology on dialyzer transport and provide important insights into the effective clinical performance observed with the highly asymmetric ATA™ dialyzers.