Tangential flow microfiltration and ultrafiltration for human influenza A virus concentration and purification

Abstract

Large scale purification of viruses and viral vectors for gene therapy applications and viral vaccines is a major separation challenge. Here tangential flow microfiltration and ultrafiltration using flat sheet membranes has been investigated for concentration of human influenza A virus. Ultrafiltration membranes with molecular weight cutoffs of 100 and 300 kDa as well as 0.1, 0.2 and 0.45 microm microfiltration membranes have been tested. The results indicate that use of 300 kDa membranes not only concentrate the virus particles but also lead to a significant removal of host cell proteins and DNA in the permeate. Tangential flow filtration may be used to fractionate virus particles. Human influenza A virus particles are spherical with an average size of 100 nm. Use of a 0.1 microm membrane leads to passage of virus particles less than 100 nm into the permeate and an increase of larger particles in the retentate. These results suggest that control of the transmembrane pressure, membrane pore size and pore size distribution could enable isolation of intact virus particles from damaged virions. Isolation of the virus particles of interest from viral fragments and other particulate matter could result in simplification of subsequent purification steps. Larger pore size membranes such as 0.45 microm that allow the passage of all virus particles may be used to remove host cell fragments. In addition virus particles attached to these fragments will be removed. Careful selection of membrane morphology and operating conditions will be essential in order to maximize the benefit of tangential flow filtration steps in the purification of viral products from cell cultures.

Harvesting and concentration of human influenza A virus produced in serum‐free mammalian cell culture for the production of vaccines

“…Here, a 100 kDa flat sheet module made of polyethersulfone was employed. Using similar virus starting material, Wickramasinghe et al (2005) investigated the suitability of cross-flow flat sheet ultra-and microfiltration modules with cut-offs ranging from 100 kDa to 0.45 mm in more detail.…”

Atomic Force Microscopy in Detection of Viruses

“…Also, surface morphology is characterized by the presence of large nanoparticles, typically 120 to 250 nm wide at the base and 50 to 100 nm tall. Now, it is known that the influenza virus is a quasi-spherical object, approximately of 100 to 120 nm in diameter (Wickramasinghe, Kalbfuss et al 2005). …”

DNA Depletion by Precipitation in the Purification of Cell Culture‐Derived Influenza Vaccines

“…Concentrations of double-stranded DNA were estimated to be similar to Wickramasinghe et al [26]. Briefly, samples of 200 lL were mixed with 50 lL of Pico Green (Cat.…”

“…Tangential-flow ultrafiltration was selected for the concentration of influenza virions, since this method is well established and had already been described for a number of viruses [9,26,43,45,46,[59][60][61][62]. Diafiltration, following the concentration step, was added as a replacement for SEC that had been used in a previous study [10].…”