Live virus vaccine (LVV) purification, employing chromatography, can be challenged by low binding capacities and elution yields. Alternatively, processes relying solely on enzymatic digestion steps ...and size-based membrane separations can be limited by suboptimal reduction of process related impurities and poorly scalable unit operations. Here, we demonstrate that the combination of flowthrough mode chromatography and an ultrafiltration/diafiltration (UF/DF) unit operation delivers a purification process for two different LVV candidates, V590 and Measles, expressed in adherent Vero cells. For V590, chromatography with mixed mode cation exchange resins returned final product yields of ∼50% and logarithmic reduction values (LRVs) of 1.7->3.4 and 2.5-3.0 for host cell DNA (hcDNA) and host cell proteins (HCPs), respectively. For Measles, chromatography with mixed mode anion exchange resins returned final product yields of ∼50% and LRVs of 1.6 and 2.2 for hcDNA and HCPs, respectively. For both V590 and Measles processing, the employed resins cleared a key HCP, fibronectin, which could foul the UF/DF unit operation, and thusly enabling it to further reduce HCPs and to formulate the final LVV products. This integrated purification process utilizes the complementary action of the two unit operations and its applicability across LVVs supports its consideration for their processing.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Prefilters are widely used to enhance the performance of normal flow filtration steps in bioprocessing; however little is known about the potential benefits of prefiltration in the sterile filtration ...of viral vaccines and other large particle biotherapeutics. Sterile filtration experiments were performed with a live-attenuated viral vaccine and model nanoparticle suspension, both of which have a size distribution of 100 – 400 nm. The results demonstrated that the outstanding performance of the Sartobran P sterile filter was directly due to the effective prefiltration provided by the 0.45 μm prefilter in this dual layer filter. The 0.45 μm prefilter dramatically improved particle transmission and capacity for other 0.2 μm sterile filters. The key foulants were removed primarily on the basis of size, which was confirmed using a nanoparticle tracking analyzer. Larger pore size prefilters were found to be much less effective in protecting the 0.2 μm sterile filter. The support structure of asymmetric sterile filters also provided significant prefiltration; however, some of these asymmetric membranes had size-selective pores that were too small to provide significant transmission of the viral particles. These results provided important insights into the role of the prefilter layer and membrane pore structure in enhancing the performance of the sterile filtration step for the processing of viruses and other large particle biotherapeutics.
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•Sterile filtration performed with Live Attenuated Viral vaccine (LAV).•Role of prefilter in Sartobran P dual-layer filter quantified.•Prefilter improves capacity and transmission by removing large particles.•Support structure of asymmetric membrane also provides effective prefiltration.•Combination of prefilter and sterile filter pore size determines performance.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ
Sterile filtration is used to remove microorganisms in the processing of most recombinant proteins, but there are significant challenges in applying this technology to large biotherapeutics like ...Live-Attenuated Vaccines (LAV), lipid-nanoparticles, and gene therapy agents. Previous studies have reported highly variable fouling and product retention behavior of different sterile filters in these applications, but there has been no clear understanding of the origin of these differences. This study used fluorescently-labeled 200, 300, and 400 nm polystyrene latex spheres as model particle challenges for eight commercially available, 0.2/0.22 μm pore size sterile filters, all of which were characterized using mercury intrusion porosimetry, bubble point measurements, and scanning electron microscopy. All filters showed significant fouling, causing an increase in particle retention at high throughput. The maximum transmission of the 300 nm particles varied from only 0.01 for the highly asymmetric Millipore Express® to 0.9 for the dual-layer Sartobran® P. The transmission was well correlated with the ratio of the particle diameter to the maximum pore size (determined by bubble point), with even better agreement obtained using the mean pore size evaluated from mercury intrusion porosimetry. These results provide important insights into the effects of pore size and structure on the retention characteristics of sterile filter as well as guidance on how to select the best sterile filters for processing vaccines and viruses that are >100 nm in size.
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•Nanoparticle retention evaluated for range of commercial 0.2 μm sterile filters.•Transmission of 300 nm particles varied between 1% and >90%.•Filters characterized by bubble point and mercury porometry.•Differences in transmission directly related to small differences in pore size.•Particle fouling resulted in decrease in transmission at high throughput.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ
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