Probing effects of pressure release on virus capture during virus filtration using confocal microscopy

Abstract

Virus filtration is used to ensure drug safety in the production of biotherapeutics. Several recent studies have shown a dramatic decrease in virus retention as a result of a process disruption, e.g., a transient pressure release. In this work, a novel two-label fluorescence technique was developed to probe virus capture within virus filtration membranes using confocal microscopy. Experiments were performed with Ultipor® DV20, Viresolve® Pro, and Viresolve® NFP membranes using bacteriophage φx174 as a model virus. The filters were challenged with two batches of fluorescently labeled phage: one labeled with red dye (Cy5) and one with green dye (SYBR Gold) to visualize captured phage from before and after the pressure release. The capture patterns seen in the confocal images were a strong function of the underlying membrane morphology and pore structure. The DV20 and Viresolve® NFP showed migration of previously captured phage further into the filter, consistent with the observed loss of virus retention after the pressure release. In contrast, there was no migration of captured virus in the Viresolve® Pro membranes, and these filters were also the only ones to show stable virus retention after a pressure release. The direct visualization of virus capture using the two-label fluorescence technique provides unique insights into the factors controlling the retention characteristics of virus filters with different pore structure.

Interactions between protein molecules and the virus removal membrane surface: Effects of immunoglobulin G adsorption and conformational changes on filter performance

“…Recent virus filtration studies have focused on the effects of process parameters on throughput and viral retention. Operating pressure and solution conditions have been identified as crucial parameters for virus retention. Dishari et al indicated that solution pH and ionic strength can alter the electrostatic interactions between viruses and membranes, affecting virus retention .…”

Control of antibody high and low molecular weight species by depth filtration‐based cell culture harvesting

“…In large scale biologics manufacturing, depth filters are mostly considered as mechanical entrapment device and little attention is paid to exploit adsorption mechanism for purification advantage. Adsorptive filters have also been studied to provide viral and endotoxin clearance that suggested either electrostatic adsorption or a combination of adsorption and mechanical entrapment (Dishari et al, 2015;Hou & Zaniewski, 1990;Michen et al, 2012;Tipton, Boose, Larsen, Beck, & O Brien, 2002). Adsorptive filters have also been studied to provide viral and endotoxin clearance that suggested either electrostatic adsorption or a combination of adsorption and mechanical entrapment (Dishari et al, 2015;Hou & Zaniewski, 1990;Michen et al, 2012;Tipton, Boose, Larsen, Beck, & O Brien, 2002).…”

“…Adsorptive filters have also been studied to provide viral and endotoxin clearance that suggested either electrostatic adsorption or a combination of adsorption and mechanical entrapment (Dishari et al, 2015;Hou & Zaniewski, 1990;Michen et al, 2012;Tipton, Boose, Larsen, Beck, & O Brien, 2002). Venkiteshwaran et al (Dishari et al, 2015) has found that porcine parvovirus (PPV) was cleared by X0HC and B1HC depth filters at < 2.5 mS/cm but not at 20 mS/cm, suggesting charge interaction at low conductivity; whereas xenotropic murine leukemia virus (XMuLV) was cleared at high conductivity (20 mS/cm) by X0HC, B1HC, and EKSP, suggesting strong involvement of hydrophobic interactions. It was demonstrated that not only electrostatic interactions but also hydrophobic interactions between the depth filters and DNA molecules played important roles in the adsorptive reduction of DNA.…”

Suitability of a generic virus safety evaluation for monoclonal antibody investigational new drug applications

“…An industry‐wide evaluation of small virus retentive filters also found that large enveloped viruses were reduced below the limit of detection in all 198 cases examined, providing additional rationale that viral testing only needs to be performed with smaller virus as a worst case scenario . It has been shown that virus breakthrough can occur with small virus retentive filters during long processing times . However, proper evaluation and control of process parameters and solution properties can maintain high filter flux and mitigate this risk .…”