Virus harvesting in perfusion culture: Choosing the right type of hollow fiber membrane

Abstract

The use of bioreactors coupled to membrane‐based perfusion systems enables very high cell and product concentrations in vaccine and viral vector manufacturing. Many virus particles, however, are not stable and either lose their infectivity or physically degrade resulting in significant product losses if not harvested continuously. Even hollow fiber membranes with a nominal pore size of 0.2 µm can retain much smaller virions within a bioreactor. Here, we report on a systematic study to characterize structural and physicochemical membrane properties with respect to filter fouling and harvesting of yellow fever virus (YFV; ~50 nm). In tangential flow filtration perfusion experiments, we observed that YFV retention was only marginally determined by nominal but by effective pore sizes depending on filter fouling. Evaluation of scanning electron microscope images indicated that filter fouling can be reduced significantly by choosing membranes with (i) a flat inner surface (low boundary layer thickness), (ii) a smooth material structure (reduced deposition), (iii) a high porosity (high transmembrane flux), (iv) a distinct pore size distribution (well‐defined pore selectivity), and (v) an increased fiber wall thickness (larger effective surface area). Lowest filter fouling was observed with polysulfone (PS) membranes. While the use of a small‐pore PS membrane (0.08 µm) allowed to fully retain YFV within the bioreactor, continuous product harvesting was achieved with the large‐pore PS membrane (0.34 µm). Due to the low protein rejection of the latter, this membrane type could also be of interest for other applications, that is, recombinant protein production in perfusion cultures.

Cell culture–based production of defective interfering influenza A virus particles in perfusion mode using an alternating tangential flow filtration system

“…3b–d ). For the used HFM, it is well established that virus particles of about 40–100 nm in diameter cannot pass through the membrane (Nikolay et al 2020 ). Samples taken shortly before harvesting confirmed this, since only residual virus amounts could be detected in the permeate line (Fig.…”

Cell culture-based production of defective interfering influenza A virus particles in perfusion mode using an alternating tangential flow filtration system

“…3b–d). For the used HFM it is well established that virus particles of about 40-100 nm in diameter cannot pass through the membrane (Nikolay et al 2020). Samples taken shortly before harvesting confirmed this, since only residual virus amounts could be detected in the permeate line (Fig.…”

“…Previous studies investigated different HFMs for their potential to allow continuous virus harvesting (Genzel et al 2014; Nikolay et al 2020). However, almost all tested membranes completely retained the produced virus particles, despite the nominal pore size reported was much larger than the virus diameter.…”

Process intensification for the production of yellow fever virus‐like particles as potential recombinant vaccine antigen

“…As discussed above, product retention had already been observed for virus and VLP production when typical polyethersulfone ATF cartridges with 0.2 μm pore size and 0.13 m 2 filtration area were used (Alvim et al, 2019; Coronel et al, 2019; Lavado‐García et al, 2020). In spite of using in the present work a different membrane cartridge, having the same characteristics (polysulfone membranes with 0.4 µm pore size and 0.005 m² filtration area) as the cartridge that Nikolay et al (2020b) found to be adequate for yellow fever whole virus processing, product retention did occur again.…”

“…In our previous work on zika VLP production (Alvim et al, 2019), although a perfusion run could be stably operated for 30 days at approximately 30 million cells/mL, product retention inside the bioreactor due to membrane fouling was observed. However, considering a recent report (Nikolay et al, 2020b) that showed a polysulfone hollow fiber cartridge to be highly permeable for the YFV, we decided to use a cartridge of hollow fibers having the same characteristics as reported by these authors, with the aim of avoiding fouling and product retention.…”