Virus Removal Filter Research Articles

Comparative Analysis of the Impact of Protein on Virus Retention for Different Virus Removal Filters.

The performance of virus filters is often determined by the extent of protein fouling, which can affect both filtrate flux and virus retention. However, the mechanisms governing changes in virus retention in the presence of proteins are still not well understood. The objective of this work was to examine the effect of proteins on virus retention by both asymmetric (Viresolve® NFP and Viresolve® Pro) and relatively homogeneous (Ultipor® DV20 and PegasusTM SV4) virus filtration membranes. Experiments were performed with bacteriophage ϕX174 as a model parvovirus and human serum immunoglobulin G (hIgG) as a model protein. The virus retention in 1 g/L hIgG solutions was consistently less than that in a protein-free buffer solution by between 1 to 3 logs for the different virus filters. The virus retention profiles for the two homogeneous membranes were very similar, with the virus retention being highly correlated with the extent of flux decline. Membranes prefouled with hIgG and then challenged with phages also showed much lower virus retention, demonstrating the importance of membrane fouling; the one exception was the Viresolve® Pro membrane, which showed a similar virus retention for the prefouled and pristine membranes. Experiments in which the protein was filtered after the virus challenge demonstrated that hIgG can displace previously captured viruses from within a filter. The magnitude of these effects significantly varied for the different virus filters, likely due to differences in membrane morphology, pore size distribution, and chemistry, providing important insights into the development/application of virus filtration in bioprocessing.

Virus retention during constant-flux virus filtration using the Viresolve® Pro membrane including process disruption effects

The growing interest in process intensification and continuous processing has led to concerns regarding the performance of virus removal filters at low permeate fluxes. This paper presents a quantitative analysis of virus retention by the Viresolve® Pro membrane during constant permeate flux filtration and in response to process disruptions using ΦX-174, a bacteriophage, as a model for a mammalian parvovirus. Virus retention by a single layer of Viresolve® Pro membrane was reduced at lower permeate fluxes due to the greater diffusive mobility of phage, although the expected retention for a commercial device employing two layers of membrane remained above 4-logs. The retention data were well correlated with the ΦX-174 Péclet number for filtrate flux from 5 to 100 L/m2/h. Process disruptions caused an immediate transient increase in phage transmission, although this effect was much less pronounced at low permeate flux. The experimental data for phage retention, both with and without process disruptions, were well-described using an internal polarization model with the fraction of virus remaining mobile within the filter scaling as 1/Pe2. In contrast, the fraction of previously captured virus that were re-mobilized after a process interruption was independent of the flow rate. Model equations were developed for rapid estimation of virus performance behavior with the Viresolve® Pro membrane, facilitating the design and implementation of virus filtration processes operating at constant filtrate flux.

Size-based analysis of virus removal filter fouling using fractionated protein aggregates.

Fouling by protein aggregates reduces virus removal filter performance. In the present study, we investigated the effects of different-sized protein aggregates on fouling and aggregate retention in order to better understand the fouling mechanisms. Human immunoglobulin G was denatured by heating to produce aggregates of various sizes and then fractionated by size exclusion chromatography into different-sized aggregates with a narrow size distribution. The fractionated aggregates were filtered on Planova 20N, a virus removal filter known for its stable filtration capability. Analysis of flux behavior demonstrated different flux decrease patterns for different-sized aggregates. Observation of aggregate retention by staining revealed that larger aggregates were captured closer to the inner surface of the membrane while smaller aggregates penetrated farther into the membrane. These findings demonstrate that Planova 20N has a gradient structure with decreasing pore size from the inner to the outer surface of the membrane. This structure minimizes fouling and enables stable filtration by protecting the smaller pores located closer to the outer surface from clogging by large aggregates. Applying the predominant clogging models to the present filtrations revealed that clogging behavior transitioned from complete blocking to cake filtration as filtration progressed. In this combination model, after a certain number of pores are blocked by complete blocking, newly arrived aggregates begin to accumulate on previously captured aggregates, generating cake between capture layers within the membrane. Application of the approaches described here will facilitate elucidation of membrane fouling and virus removal mechanisms.

Impact of virus filter pore size / morphology on virus retention behavior

Virus filters provide size-based removal of >99.9% of viruses and virus-like particles during biopharmaceutical processing. Here, we quantitatively evaluate the 3D pore space and virus capture behavior in two commercial virus removal filters with very different morphologies: the highly asymmetric Viresolve® Pro and the relatively homogeneous Pegasus™ SV4 virus filters. Focused ion beam (FIB) – SEM was used to reconstruct the 3D pore morphology of both virus filters. The Pegasus™ SV4 filter had pores that were, on average, significantly larger than 20 nm, which is the size of small parvovirus (as well as the average size of the pore throats near the exit of the Viresolve® Pro filter). Virus retention within each membrane was simulated using GeoDict®, which evaluates the detailed particle trajectories and capture locations through the filter. The Pegasus™ SV4 filter showed virus capture throughout a significant fraction of the filter depth, in contrast to virus capture in the Viresolve® Pro filter that occurred only in the very small pores in the 2 μm region near the filter exit. In both cases, viruses were captured in highly restrictive regions within the pore space. These results provide new insights into the impact of pore morphology on virus capture during virus removal filtration.

Analysis of filtration with virus removal filters using the characteristic form of blocking model

For virus filtration behavior not adequately described by the equations and analysis methods of the conventional four blocking models, i.e., complete blocking, standard blocking, intermediate blocking and cake filtration models, a modified approach is proposed using a generalized equation derived from the characteristic form of the blocking model with consideration of the multilayer membrane structure with pore size distribution (Manabe model). The two parameters of the characteristic form, blocking index, n, and characteristic form coefficient, k, for the filtration behavior, are easily determined by applying the modified characteristic form to experimental data. The generalized filtration equation with these two parameters shows good fit for both constant pressure filtration and constant flux filtration. For high filterability virus filter behavior, n is a negative value, and the physical meaning of a negative value can reasonably be explained in consideration of the multilayer membrane structure with pore size distribution. The assumptions of the conventional blocking model, i.e., the membrane structure consisting of single active layer with single pore size is not applicable for virus filters. Instead, the Manabe model is essential for explaining the stable filtration of the virus filter.

Effect of filtrate flux and process disruptions on virus retention by a relatively homogeneous virus removal membrane.

Recent studies have shown that virus retention by specific virus filters can be reduced at low flow rates and after process disruptions; however, the magnitude of these changes in virus retention and the underlying mechanisms controlling this behavior are still not well understood. The objective of this study was to develop a quantitative understanding of the factors controlling the virus retention behavior of a relatively homogeneous polyvinylidene fluoride virus removal filter. Data were obtained with the bacteriophage ϕX174 as a model virus. Virus retention decreased as the filtrate flux was reduced and also declined slightly over the course of the virus filtration. Virus retention immediately after a process disruption decreased by as much as a factor of 1000 (3-logs) depending on the duration and timing of the disruption. The experimental results were well-described using an internal polarization model that accounts for accumulation and release of virus during the filtration / disruption, with the key model parameters dependent on the filtrate flux. These results provide important insights into the factors controlling the virus retention behavior as well as guidelines for the effective use of virus removal filters in bioprocessing.

Effect of operating pressure on protein fouling during constant-pressure virus removal filtration

Virus removal filtration provides a robust size-based removal of enveloped and non-enveloped viruses in the production of monoclonal antibodies and serum-derived products. The filtrate flux in virus removal filtration is typically dominated by membrane fouling associated with the therapeutic protein product, but very few studies have examined the effect of operating pressure on the fouling rate/mechanisms or the virus filter capacity. Filtration experiments were performed using Viresolve® Pro virus removal filters with human serum Immunoglobulin G (hIgG) as a model protein over a range of operating pressures from 10 to 60 psi. The extent of fouling (evaluated in terms of the overall resistance at a given throughput) decreased with increasing operating pressure up to about 30 psi, while the filter capacity (evaluated using the Vmax model) increased with increasing pressure. The flux decline behavior was shown to be in good agreement with the combined complete pore blockage and cake filtration model, with the pore blockage parameter decreasing with increasing pressure, while the cake filtration parameter increased with increasing pressure. Confocal microscopy was used to identify the location and extent of protein deposition within the membrane cross-section, providing further support for the effects of pressure on both pore blockage and cake resistance. These results provide important insights into the factors controlling protein fouling during virus removal filtration and the influence of operating pressure on the flux decline and filter capacity.

FIB-SEM tomography reveals the nanoscale 3D morphology of virus removal filters

Virus removal filters are designed to ensure very high levels of removal of viruses and virus-like particles during bioprocessing, but the performance of some virus removal filters can be compromised at high-throughputs, after process disruptions, or during operation at low pressures. Several studies have hypothesized that the different behavior is due to differences in underlying pore morphology, but current techniques are limited to examining the 2D pore structure. Here, we use the combination of a focused ion beam and scanning electron microscopy (FIB-SEM) to obtain 3D reconstructions of the pore structure of the asymmetric Viresolve® Pro virus removal filter. Images were obtained through a 3 μm section into the membrane starting at the size-selective skin. The membrane porosity decreases from 41 to 17% as one approaches the filter exit. Model simulations based on flow through the full 3D pore reconstruction show 100% virus retention, with all virus particles captured at least 400 nm from the filter exit. A pore-network model was developed from the reconstructions and used to evaluate the body and throat size distribution and pore interconnectivity. The number of throats is approximately twice the number of bodies, with an average throat size of 21 nm within the selective skin. The pore interconnectivity remains relatively constant at a value of 4 throats per body. These results provide insights into the underlying pore structure of the Viresolve® Pro virus removal filter as well as a general framework for characterizing the 3D pore space in nanoporous membranes.

Cationic Nanocellulose as Promising Candidate for Filtration Material of COVID-19: A Perspective

The threat of the novel coronavirus (COVID-19) pandemic is worrying as millions of people suffered from this outbreak. The COVID-19 can be airborne by attaching to human nasal or saliva secretion of an infected person or suspended fine particulates in the air. Therefore, in order to minimize the risks associated with this pandemic, an efficient, robust and affordable air‐borne virus removal filters are highly demanded for prevention of spreading viruses in hospitals, transportation hubs, schools, and/or other venues with high human turn‐over. Respirators such as N95, N99 and N100 as well as surgical masks have been widely used. To date, there is no filter standards or special filter technologies tailored for effectively adsorbing the airborne viruses. Studies had shown the electrostatic fibers were capable to entrap the negatively charged viruses including COVID-19. Researchers believed that the positive surface charge of filtration material is an important key to efficiently adsorb the negatively charged viruses. Nanocellulose has emerged as a new class of biobased material with promising potential application in the filtration of viruses. Nanocellulose which is uniform in diameter and has excellent nanofibrillar morphology. To the best of our knowledge, lack of study is done to determine the efficiency of cationic nanocellulose as filtration material of COVID-19.

Virus filter scalability: Demonstration of consistent viral clearance across laboratory and manufacturing scales

Virus removal filtration processes in biopharmaceutical manufacturing are developed, optimized and validated for viral clearance using laboratory scale filters. Thus, the scalability of these filters is critical for accurately extrapolating filtration performance and reliably extending viral clearance to manufacturing scale. Virus removal filter manufacturers generally validate scalability of filtration performance based on various filtration parameters, and virus removal capability is extended to manufacturing scale filters using inert, size-appropriate particles such as gold nanoparticles to avoid the risks associated with using mammalian viruses in large feed volumes. In this study, we use bacteriophage PP7 as a parvovirus model to directly demonstrate viral clearance on Planova™ BioEX virus removal filters across all scales, including manufacturing scale. Filters with hollow fibers from three spinning series with filter sizes ranging from 0.0003 to 4.0 m2 were tested for virus removal, flux, and protein recovery performance using BSA spiked with PP7. Complete viral clearance was observed across all filter sizes with PP7 LRV of ≥4.7 or higher. Flux and protein recovery were also consistent. These results demonstrate the scalability of filtration performance and consistent virus removal at all sizes, supporting the use of laboratory scale filters to validate viral clearance at manufacturing scales.

Direct visualization of virus removal process in hollow fiber membrane using an optical microscope

Virus removal filters developed for the decontamination of small viruses from biotherapeutic products are widely used in basic research and critical step for drug production due to their long-established quality and robust performance. A variety of imaging techniques have been employed to elucidate the mechanism(s) by which viruses are effectively captured by filter membranes, but they are limited to ‘static’ imaging. Here, we propose a novel method for detailed monitoring of ‘dynamic process’ of virus capture; specifically, direct examination of biomolecules during filtration under an ultra-stable optical microscope. Samples were fluorescently labeled and infused into a single hollow fiber membrane comprising cuprammonium regenerated-cellulose (Planova 20N). While proteins were able to pass through the membrane, virus-like particles (VLP) accumulated stably in a defined region of the membrane. After injecting the small amount of sample into the fiber membrane, the real-time process of trapping VLP in the membrane was quantified beyond the diffraction limit. The method presented here serves as a preliminary basis for determining optimum filtration conditions, and provides new insights into the structure of novel fiber membranes.

Introduction and clearance of beta-glucan in the downstream processing of monoclonal antibodies.

β‐Glucan process‐related impurities can be introduced into biopharmaceutical products via upstream or downstream processing or via excipients. This study obtained a comprehensive process‐mapping dataset for five monoclonal antibodies to assess β‐glucan introduction and clearance during development and production runs at various scales. Overall, 198 data points were available for analysis. The greatest β‐glucan concentrations were found in the depth‐filtration filtrate (37–2,745 pg/ml). Load volume correlated with β‐glucan concentration in the filtrate, whereas flush volume was of secondary importance. Cation‐exchange chromatography significantly cleared β‐glucans. Furthermore, β‐glucan leaching from the Planova 20N virus removal filter was reduced by increasing the flush volume (1 vs. 10 L/m2). β‐glucan concentrations after filter flush with 10 L/m2 were consistently <10 pg/ml. No or only limited β‐glucan clearance was attained via ultrafiltration/diafiltration (UF/DF). However, during the first run with monoclonal antibody (mAb) 4, β‐glucan concentration in the UF/DF retentate was 10.8 pg/mg, potentially due to β‐glucan leaching from the first run with a regenerated cellulose membrane. Overall, β‐glucan levels in the final mAb drug substance were 1–12 pg/mg. Assuming high doses of 1,000–5,000 mg, a β‐glucan contamination at 20 pg/mg would translate to 20–100 ng/dose, which is below the previously suggested threshold for product safety (≤500 ng/dose).

Two-Step Size-Exclusion Nanofiltration of Prothrombin Complex Concentrate Using Nanocellulose-Based Filter Paper.

Coagulation Factor IX-rich protrhombin complex concentrate (FIX-PCC) is a therapeutic biologic product that consists of a mixture of several human plasma-derived proteins, useful for treating hemophilia B. Due to its complex composition, FIX-PCC is very challenging to bioprocess through virus removing nanofilters in order to ensure its biosafety. This article describes a two-step filtration process of FIX-PCC using a nanocellulose-based filter paper with tailored porosity. The filters were characterized with scanning electron microscopy (SEM), cryoporometry with differential scanning calorimetry, and nitrogen gas sorption. Furthermore, in order to probe the filter’s cut-off size rejection threshold, removal of small- and large-size model viruses, i.e., ΦX174 (28 nm) and PR772 (70 nm), was evaluated. The feed, pre-filtrate, and permeate solutions were characterized with mass-spectrometric proteomic analysis, dynamic light scattering (DLS), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and analytical size-exclusion high-performance liquid chromatography (SEHPLC). By sequential filtration through 11 μm pre-filter and 33 μm virus removal filter paper, it was possible to achieve high product throughput and high virus removal capacity. The presented approach could potentially be applied for bioprocessing other protein-based drugs.

Asahi Kasei Medical builds new spinning plant for Planova filters

Asahi Kasei Medical has completed a new plant in Nobeoka, Miyazaki, Japan for the spinning of cellulose hollow-fiber membranes for Planova™ virus removal filters.

Microscopic visualization of virus removal by dedicated filters used in biopharmaceutical processing: Impact of membrane structure and localization of captured virus particles.

Virus filtration with nanometer size exclusion membranes (“nanofiltration”) is effective for removing infectious agents from biopharmaceuticals. While the virus removal capability of virus removal filters is typically evaluated based on calculation of logarithmic reduction value (LRV) of virus infectivity, knowledge of the exact mechanism(s) of virus retention remains limited. Here, human parvovirus B19 (B19V), a small virus (18–26 nm), was spiked into therapeutic plasma protein solutions and filtered through Planova™ 15N and 20N filters in scaled‐down manufacturing processes. Observation of the gross structure of the Planova hollow fiber membranes by transmission electron microscopy (TEM) revealed Planova filter microporous membranes to have a rough inner, a dense middle and a rough outer layer. Of these three layers, the dense middle layer was clearly identified as the most functionally critical for effective capture of B19V. Planova filtration of protein solution containing B19V resulted in a distribution peak in the dense middle layer with an LRV >4, demonstrating effectiveness of the filtration step. This is the first report to simultaneously analyze the gross structure of a virus removal filter and visualize virus entrapment during a filtration process conducted under actual manufacturing conditions. The methodologies developed in this study demonstrate that the virus removal capability of the filtration process can be linked to the gross physical filter structure, contributing to better understanding of virus trapping mechanisms and helping the development of more reliable and robust virus filtration processes in the manufacture of biologicals.

Virus removal filtration of chemically defined Chinese Hamster Ovary cells medium with nanocellulose-based size exclusion filter

Sterility of bioreactors in biotherapeutic processing remains a significant challenge. Virus removal size-exclusion filtration is a robust and highly efficient approach to remove viruses. This article investigates the virus removal capacity of nanocellulose-based filter for upstream bioprocessing of chemically defined Chinese hamster ovary (CHO) cells medium containing Pluronic F-68 (PowerCHO™, Lonza) and supplemented with insulin-transferrin-selenium (ITS) at varying process parameters. Virus retention was assessed by spiking ITS-supplemented PowerCHO™ medium with small-size ΦX174 phage (28 nm) as a surrogate for mammalian parvoviruses. The nanocellulose-based size exclusion filter showed high virus retention capacity (over 4 log10) and high flow rates (around 180 L m−2 h−1). The filter had no impact on ITS supplements during filtration. It was further shown that the filtered PowerCHO™ medium supported cell culture growth with no impact on cell viability, morphology, and confluence. The results of this work show new opportunities in developing cost-efficient virus removal filters for upstream bioprocessing.

Comparative Analysis of Dry and Wet Porometry Methods for Characterization of Regular and Cross-Linked Virus Removal Filter Papers.

Pore-size distribution (PSD) is the most critical parameter for size-exclusion virus removal filters. Yet, different dry- and wet-state porometry methods yield different pore-size values. The goal of this work is to conduct comparative analysis of nitrogen gas sorption (NGSP), liquid-liquid and cryoporometry with differential scanning calorimetry (CP-DSC) methods with respect to characterization of regular and cross-linked virus removal filter paper based on cellulose nanofibers, i.e. the mille-feuille filter. The filters were further characterized with atomic force and scanning electron microscopy. Finally, the removal of the worst-case model virus, i.e. minute virus of mice (MVM; 20 nm, nonenveloped parvovirus) was evaluated. The results revealed that there is no difference of the obtained PSDs between the wet methods, i.e. DSC and liquid-liquid porometry (LLP), as well as no difference between the regular and cross-linked filters regardless of method. MVM filtration at different trans membrane pressure (TMP) revealed strong dependence of the virus removal capability on applied pressure. It was further observed that cross-linking filters showed enhanced virus removal, especially at lower TMP. In all, the results of this study highlight the complex nature of virus capture in size-exclusion filters.

Growth media filtration using nanocellulose-based virus removal filter for upstream biopharmaceutical processing

The feasibility of using nanocellulose-based mille-feuille filter paper for upstream applications in serum-free growth media filtration, i.e. Dulbecco's modified Eagle's medium (DMEM) and Luria-Bertani medium (LBM), was tested. The filter performance with respect to ΦΧ174 bacteriophage (28 nm) removal as a model small-size virus was characterized for filters of varying thicknesses, i.e. 11 and 33 µm, at varying operating pressures, i.e. 1 and 3 bar. The filters demonstrated generally good model small-size virus removal properties with LRV ≥ 5, especially for 33 µm filters. The 33 µm filters were more robust and exhibited better virus removal and throughput properties than 11 µm filters, although their flux was generally lower. The performance of the 33 and 11 µm nanocellulose-based filter papers was further verified for upscaled bioprocessing by 10-fold increase in the loading volume. The results of the present work show that the 33 µm nanocellulose-based filter paper could be an interesting alternative for large volume cell culture medium filtration during upstream bioprocessing.

Significance of Brownian Motion for Nanoparticle and Virus Capture in Nanocellulose-Based Filter Paper.

Pressure-dependent breakthrough of nanobioparticles in filtration was observed and it was related to depend on both convective forces due to flow and diffusion as a result of Brownian motion. The aim of this work was to investigate the significance of Brownian motion on nanoparticle and virus capture in a nanocellulose-based virus removal filter paper through theoretical modeling and filtration experiments. Local flow velocities in the pores of the filter paper were modeled through two different approaches (i.e., with the Hagen–Poiseuille equation) and by evaluating the superficial linear flow velocity through the filter. Simulations by solving the Langevin equation for 5 nm gold particles and 28 nm ΦX174 bacteriophages showed that hydrodynamic constraint is favored for larger particles. Filtration of gold nanoparticles showed no difference in retention for the investigated fluxes, as predicted by the modeling of local flow velocities. Filtration of ΦX174 bacteriophages exhibited a higher retention at higher filtration pressure, which was predicted to some extent by the Hagen–Poiseuille equation but not by evaluation of the superficial linear velocity. In all, the hydrodynamic theory was shown able to explain some of the observations during filtration.

High-Performance Virus Removal Filter Paper for Drinking Water Purification.

Access to drinking water is one of the greatest global challenges today. In this study, the virus removal properties of mille‐feuille nanocellulose‐based filter papers of varying thicknesses from simulated waste water (SWW) matrix are evaluated for drinking water purification applications. Filtrations of standard SWW dispersions at various total suspended solid (TSS) content are performed, including spiking tests with 30 nm surrogate latex particles and 28 nm ΦX174 bacteriophages. Filter papers of thicknesses 9 and 29 µm are used, and the filtrations are performed at two different operational pressures, i.e., 1 and 3 bar. The presented data using SWW matrix show, for the first time, that a filter paper made from 100% nanocellulose has the capacity to efficiently remove even the smallest viruses, i.e., up to 99.9980–99.9995% efficiency, at industrially relevant flow rates, i.e., 60–500 L m−2 h−1, and low fouling, i.e., V max > 103–104 L m−2. The filter paper presented in this work shows great promise for the development of robust, affordable, and sustainable water purification systems.