Virus Filtration Research Articles

Single particle tracking reveals through-membrane diffusion of bacteriophage during process disruption of virus filtration

In the biopharmaceutical industry, virus filters are crucial for ensuring the removal of endogenous and adventitious viruses as part of the viral clearance strategy. Although traditionally described as a size-exclusion mechanism, virus retention has a process-dependent nature where challenging conditions, such as process disruptions, may compromise membrane retention and significantly increase virus filtrate concentrations. The detailed mechanisms underlying this loss of retention are challenging to determine using traditional breakthrough experiments. In this work, single particle tracking and kinetic simulations were employed to connect individual particle behavior to the observed macroscopic losses in virus retention. Our experiments, using fluorescently labeled ΦX174 bacteriophage as a model parvovirus, replicated conditions representative of process disruptions within the Pegasus SV4, a homogeneous polymeric virus filtration membrane. During flow, phage particles retained were trapped within relatively large cavity spaces that had downstream constrictions aligned with the flow direction; the trapped particles were dynamic and exhibited significant intra-cavity motion. Upon flow stoppage, particles escaped from these retention locations rapidly, with approximately 90 % of previously trapped particles being remobilized for process disruption times ranging from 2 to 10 min, suggesting that local cavity escape had reached saturation at these timescales. Diffusion experiments within the membrane revealed isotropic and Fickian motion, hindered by more than an order of magnitude compared to diffusion in unconfined liquid. Despite the reduced mobility within the membrane, the substantial diffusion coefficient of 4.19 ± 0.06 μm2/s indicated that virus particles could travel tortuous but non-retentive pathways through the membrane on length scales equal to or greater than the membrane thickness during a disruption event. A 1D kinetic Monte-Carlo simulation successfully connected single-particle behavior to macroscopically observed virus release, indicating that significant diffusive release into the filtrate can occur even without the resumption of flow. This work provides crucial insights into the retention behavior of homogeneous membranes during periods of disruption, enabling the design of more robust mitigation strategies and filter designs.

Microscale insights into deep bed membrane filtration: Influence of internal surface roughness

Deep bed filtration is widely applied in bioprocessing, virus filtration, and water purification to remove small impurities, such as particles or virus fragments. However, more needs to be understood about the parameters that influence particle capture and deposition. Detailed simulations and microfluidic filtration experiments with straight pores have been widely investigated, yet realistic membrane porosity features such as pore gradients and roughness have not been addressed in detail. The internal membrane morphology is assumed to have a strong effect on filtration performance. Therefore, this study introduces a new microfluidic membrane mimicking device (MMD) and methodology that analyzes spatio-temporal particle collections in a deep bed filtration MMD with gradually decreasing pore size toward the retentate side. The design allows us to systematically tailor an internal filter surface roughness to investigate its influence on differently sized soft particles and the consequences for filtration performance, in particular, particle capture. Optical investigation and quantitative evaluation show enhanced particle–pillar interactions for increased roughness. This results in a reduced penetration depth and reduced particle breakthrough. The modes of capture or filtration phenomena, such as pore blocking, bridging, or dendrite formation, differ significantly between smooth and rough membrane filter structures. In the case of rough inner filter surfaces, those phenomena lead to a less distinct decrease in volumetric flow, allowing a longer filtration process. The microfluidic study offers a detailed investigation of how microscale phenomena influence the filtration process. The results provide a fundamental understanding of microscale filtration effects based on roughness and, hence, motivate a tailoring of the internal membrane filter surface according to the filtration problem at hand.

Enhancing virus inhibition in track-etched membranes through surface modification with silver nanoparticles and curcumin

The present work reports on the use of silver nanoparticles (AgNPs) and curcumin immobilized on the surface of microfiltration track-etched membranes (mTMs) as antiviral filtration materials. AgNPs were synthesized through the direct reduction of silver nitrate by sodium borohydride in the presence of a stabilizing agent. The synthesized nanoparticles and curcumin were immobilized on polyethyleneimine (PEI)-modified mTMs through a static sorption process. TEM, SEM, UV–vis. spectroscopy and luminescence measurements revealed a uniform distribution of AgNPs and confirmed the presence of curcumin on the membrane surface. The resulting TM-PEI-AgNPs-PVP and TM-PEI-Curcumin hybrid membranes were employed for the filtration of HSV-1, VSV, IAV, and EMCV viruses. While both membrane types exhibited insignificant inhibition for the latter two, they achieved 95 and 99 % inhibition for HSV-1 and VSV, respectively. These findings suggest that hybrid membranes with immobilized AgNPs and curcumin can have potential as antiviral filtration materials.

Virus inactivation using an electrically conducting virus filter in biopharmaceutical manufacturing process

Biopharmaceutical manufacturing processes using mammalian cells or plasma carry the risk of viral contamination. To mitigate these risks, it is essential to ensure viral clearance during the downstream process. Virus-retentive filters are used for size-based virus filtration, offering robust viral removal of more than 99.99%. However, virus breakthroughs have also been reported during virus filtration under certain conditions. In addition, these virus-retentive filters are disposable to ensure the safety of bioproducts, leading to significant costs and environmental concerns. In this study, innovative electrically conducting virus filters were fabricated using free-standing carbon veils (CV) and used to achieve additional virus inactivation after filtration. The viruses were captured in a CV-assisted virus filter, which was electrically heated using direct current to inactivate the viruses. This electrically conducting virus filter can inactivate viruses and can be reused up to five times. These results demonstrate that electrical conduction through electrical conducting damaged the phage capsid and eliminated the RNA genome, leading to bacteriophage inactivation. Moreover, it was confirmed that the electrically conducting virus filter could be reused up to five times without any changes to its physical or chemical structure. This study contributes to the reduction of process costs and environmental impacts by enabling the reuse of virus filters and enhancing the safety of the virus filtration process by preventing undesired virus breakthroughs.

Transport and clogging dynamics of flexible rods in pore constrictions.

The transport and clogging behavior of flexible particles in confined flows is a complex interplay between elastic and hydrodynamic forces and wall interactions. While the motion of non-spherical particles in unbounded flows is well understood, their behavior in confined spaces remains less explored. This study introduces a coupled computational fluid dynamics-discrete element method (CFD-DEM) approach to investigate the transport and clogging dynamics of flexible rod-shaped particles in confined pore constrictions. The spatio-temporal analysis reveals the influence of the rod's initial conditions and flexibility on its transport dynamics through a pore constriction. The simulation results demonstrate an increase in the lateral drift of the rod upon exiting the pore that can be scaled with channel height confinement. The clogging dynamics are explored based on hydrodynamic and mechanical forces, unveiling conditions for mechanical clogging through sieving. The developed method allows for the deconvolution of the forces that contribute to particle trajectories in confined flow, which is highly relevant in particle separation processes, fibrous-shaped virus filtration, biological flows, and related applications. The method is embedded into the open-source CFDEM framework, facilitating future extensions to explore multiple particle dynamics, intermolecular forces, external influences, and complex geometries.

Impact of State Intervention During the Pandemic Crisis on the Implementation of Nanotechnological Innovations in the Czech Republic

The article deals with the issue of state interventions during the pandemic in the Czech Republic and the role of nanotechnology, which played an important role in increasing the production capacity of personal protective equipment. Nanotechnology has been rarely used in the long term for the filtration of bacteria and viruses due to its relatively high cost compared to conventional technologies that are commonly used to filter bacteria and viruses. Due to the positive demand shock and therefore the increased price, nanofibres became much more widely used in respirators and hoods. However, the global shortage of protective equipment, in addition to the high demand, also caused government intervention. These may have adversely affected the potential for global implementation of nanofibres in respirators. Based on empirical research, the aim of this paper is to identify the innovation environment during the pandemic and the interventions that disrupted the innovation environment in nanotechnology.

Optimizing virus filtration for continuous processing using serial filtration at high area ratio.

Compared to batch operation, continuous bioprocessing can offer numerous advantages, including increased productivity, improved process control, reduced footprint, and increased flexibility. However, integration of traditional batch operations into a connected process can be challenging. In contrast to batch operations run at constant pressure or high flux, virus filtration in continuous processes may be operated at very low flux. This change in operating conditions may reduce the viral retention performance of the filter which has inhibited adoption of truly continuous virus filtration. To overcome this limitation, a novel approach is described that utilizes serial virus filtration, with a high area ratio between first to second stage filters, to achieve virus retention targets. In this study, virus filters were operated continuously (except for planned process interruptions) for 200 h in a serial configuration at a first to second stage filter area ratio of 13:1 and at a first stage flux of 5 L/m2/h. While the minute virus of mice (MVM) retention performance of the first stage filter was about 4 log reduction value (LRV), there was no virus detected in the second stage filtrate, translating to an MVM LRV across the filtration train of ≥6.7. The second stage filter was the dominant flow resistance at the start of the run but, as it was protected from foulants by the first stage filter, it suffered minimal fouling and the life of the filter train was controlled by the first stage. A theoretical case study projected that continuous virus filtration using serial configuration at high area ratio would have about 30% longer filter changeout time, 14% higher productivity, and virus retention nearly sixLRV greater than single stage operation. The findings of this research are expected to provide valuable insights into optimizing virus filtration in continuous bioprocessing.

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.

Fiber-in-Tube Electrifiable Structure for Virus Filtration Self-Generated Static Electricity by Vibration/Sound.

Fiber has been considered as an ideal material for virus insulation due to the readily available electrostatic adsorption. However, restricted by the electrostatic attenuation and filtration performance decline, their long-lasting applications are unable to satisfy the requirements of medical protective equipment for major medical and health emergencies such as global epidemics, which results in both a waste of resources and environmental pollution. We overcame these issues by constructing a fiber-in-tube structure, achieving the robust reusability of fibrous membranes. Core fibers within the hollow could form generators with tube walls of shell fibers to provide persistent, renewable static electricity via piezoelectricity and triboelectricity. The PM0.3 insulation efficiency achieved 98% even after 72 h of humidity and heat aging, through beating and acoustic waves, which is greatly improved compared with that of traditional nonwoven fabric (∼10% insulation). A mask spun with our fiber also has a low breathing resistance (differential pressure <24.4 Pa/cm2). We offer an approach to enrich multifunctional fiber for developing electrifiable filters, which make the fiber-in-tube filtration membrane able to durably maintain a higher level of protective performance to reduce the replacement and provide a new train of thought for the preparation of other high-performance protective products.

Residence time distribution in continuous virus filtration.

Regulatory authorities recommend using residence time distribution (RTD) to address material traceability in continuous manufacturing. Continuous virus filtration is an essential but poorly understood step in biologics manufacturing in respect to fluid dynamics and scale-up. Here we describe a model that considers nonideal mixing and film resistance for RTD prediction in continuous virus filtration, and its experimental validation using the inert tracer NaNO3. The model was successfully calibrated through pulse injection experiments, yielding good agreement between model prediction and experiment ( 0.90). The model enabled the prediction of RTD with variations-for example, in injection volumes, flow rates, tracer concentrations, and filter surface areas-and was validated using stepwise experiments and combined stepwise and pulse injection experiments. All validation experiments achieved 0.97. Notably, if the process includes a porous material-such as a porous chromatography material, ultrafilter, or virus filter-it must be considered whether the molecule size affects the RTD, as tracers with different sizes may penetrate the pore space differently. Calibration of the model with NaNO3 enabled extrapolation to RTD of recombinant antibodies, which will promote significant savings in antibody consumption. This RTD model is ready for further application in end-to-end integrated continuous downstream processes, such as addressing material traceability during continuous virus filtration processes.

Leveraging bioanalytical characterization of fractionated monoclonal antibody pools to identify aggregation-prone and less filterable proteoforms during virus filtration.

Monoclonal antibodies (mAbs) are an essential class of biotherapeutics. A platform process is used for mAb development to ensure clinically safe and stable molecules. Regulatory authorities ensure that mAb production processes include sufficient viral clearance steps to achieve less than one virus particle per million doses of product. Virus filtration is used for size-based removal of enveloped and nonenveloped viruses during downstream processing of mAbs. Process development in mAb purification relies on empirical approaches and often includes adsorptive prefiltration to mitigate virus filter fouling. Opportunities for molecular-level prediction of mAb filterability are needed to plug the existing knowledge gap in downstream processing. A molecular-level approach to understanding the factors influencing mAb filterability may reduce process development time, material loss, and processing costs due to oversized virus filters. In this work, pH step gradient fractionation was applied on polished bulk mAb feed to obtain concentrated pools of fractionated mAb variants. Biophysical properties and quality attributes of fractionated pools, including oligomeric state (size), isoelectric point profile, diffusion interaction parameters, and glycoform profile, were determined using bioanalytical methods. Filterability (loading and throughput) of fractionated pools were evaluated. Statistical methods were used to obtain correlations between quality attributes of mAb fractions and filterability on the Viresolve Pro virus filter.

Evaluating viral filtration efficiency in non-centralized air-conditioning systems: a novel approach grounded in medical experimental data

This study enhances the understanding of virus removal efficiency by filters, crucial for predicting respiratory disease transmission risk and guiding outbreak prevention. Current research often neglects the survival behaviour of pathogens. Addressing this, our research develops a novel approach anchored in medical experimental data. A mathematical model was established to depict the dynamic changes in particulate concentration within non-centralized air-conditioned rooms, considering indoor and outdoor concentrations, natural ventilation, and filter dynamics. The model’s reliability was affirmed through particle and aerosolized pathogen filtration experiments, showing an R 2 value over 0.99, indicating strong agreement with empirical data. Analysis of filter operation, both with and without infection sources during epidemics, reveals that the use of split air conditioners and filters significantly reduces pathogen concentration. Pathogens trapped in filters undergo natural decay, considerably lowering transmission risk compared to airborne states. This research offers a scientifically grounded, precise methodology for evaluating respiratory disease transmission risks, contributing substantially to public health management.

Design and validation of a wind tunnel for viral aerosol filtration testing

Design and validation of a wind tunnel for viral aerosol filtration testing

Proceedings of the 2023 Viral Clearance Symposium, Session 7: Up- and Downstream Virus Retentive Filtration.

Session 7 of the 2023 Viral Clearance Symposium reviewed progresses in virus retentive filtrations applied to both upstream and downstream processing. Upstream topics included investigations and applications of media viral filtration for upstream cell culture viral risk mitigation. Downstream topics included evaluation of viral breakthrough in continuous processing using surrogate particles and demonstration of extensive viral filtration cycling with flow interruptions and long duration in connected process. Reuse of viral filters with proposed procedures was successfully demonstrated amid the supply chain challenge encountered during the pandemic. Discussions and additional considerations for the topics were also provided.

Quaternary ammonium salt coated air filter for bioaerosol removal from building indoor air

Developing air filters with biocidal ability is important to protect the public from infectious respiratory diseases. A simple spray-coating approach was devised to fabricate antimicrobial air filters to remove bioaerosols. The commercial antimicrobial agent Goldshield 75 was coated on the air filters through covalent immobilization, endowing the fabricated filter with long-lasting biocidal ability. All coated filters significantly inhibited both Gram-positive bacteria (Micrococcus luteus) and Gram-negative bacteria (Escherichia coli). The antibacterial ability of the coated filters is similar to the commercial AeraSafe antibacterial filter. The coated filter showed over 99.9 % antibacterial efficiency 3 months after the application of the coating. Both bacterial and virus filtration efficiencies of coated charged polypropylene filter were higher than 99.9 %. The coating did not have much effect on the NaCl aerosol filtration efficiency of the filters. This simple spray-coating strategy is a practical method for producing antimicrobial air filters for the prevention of infectious respiratory diseases.

Proceedings of the 2023 Viral Clearance Symposium, Session 2: Viral Clearance Strategy and Case Studies.

This session deals with the rational design of viral clearance studies for biopharmaceuticals including recombinant proteins such as monoclonal antibodies and, as new in scope of the symposium, also viral clearance for adeno-associated viral (AAV) vectors. For recombinant proteins, large datasets were accumulated over the last decades and are intended to be used for accelerated product process development and streamlining of viral clearance studies. How to utilize prior knowledge in viral clearance validation and how it can be used in a risk assessment tool to decide whether additional virus clearance studies are necessary during product development is being addressed by three of the presentations of this session. This also includes an a priori intended design and generation of validation data for a new kind of detergent such as CG-110, to build up a platform dataset to be used as prior knowledge in future marketing application. Another presentation investigates the virus removal mechanism of a newly developed hydrophobic interaction chromatography (HIC) resin and demonstrates for highly hydrophobic antibodies appropriate reduction for a retrovirus and impurities in a defined process range in contrast to the moderate to poor virus reduction of recent HIC resins. The last two presentations deal with virus clearance approaches for AAV, which will become mandatory with approval of the ICH Q5A revision. Appropriate virus removal and virus inactivation procedures can be implemented into the manufacturing processes of AAV vectors including viral filtration, viral inactivation (e.g., heat inactivation), affinity chromatography, and anion-exchange chromatography with which it seems possible to achieve a good clearance for helper and also adventitious viruses. The heat treatment step can be even a robust step for adenovirus helper inactivation for AAV products when product characteristics and process conditions are understood.

Effect of pH, NaCl concentration, and mAb concentration of feed solution on the filterability of Planova™ 20N and Planova™ BioEX.

Virus filtration is one of the most important steps in ensuring viral safety during the purification of monoclonal antibodies (mAbs) and other biotherapeutics derived from mammalian cell cultures. Regarding the various virus retentive filters, including Planova filters, a great deal of data has been reported on the virus retention capability and its mechanism. Along with the virus retention capability, filterability is a key performance indicator for designing a robust and high-throughput virus filtration step. In order to obtain higher filterability, optimization of the feed solution conditions, and filter selection is essential; however, limited data are available regarding the filtration characteristics of Planova filters. Furthermore, for Planova 20N and Planova BioEX, the virus retention characteristics were reported to differ due to their respective membrane materials and layer structures. Whether these filters differ in their filtration characteristics is an interesting question, but no comparative evaluations have been reported. In this study, the filterability of the two filters was investigated and compared using 15 feed mAb solutions of a single mAb selected by design of experiments with different combinations of pH, NaCl concentration, and mAb concentration. The filterability of Planova 20N was affected not only by the feed solution viscosity, but also by the mAb aggregate content of the feed mAb solution and mAb-membrane electrostatic interactions. In contrast, the filterability of Planova BioEX decreased under some buffer conditions. These findings and the established design spaces of these filters provide valuable insights into the process optimization of virus filtration.

Adapting virus filtration to continuous processing: Effects of product and process variability on filtration performance.

Virus filtration (VF) is an important unit operation in the manufacture of biotherapeutics that provides robust removal of potential virus contaminants. Small virus removal can be impacted by the low operating pressures and potential depressurization events that are often associated with continuous operations where increased operational flexibility for higher loading at low flux and low pressure is required. In this study, we evaluated the impact of low flux (7 LMH) and pressure interruptions on minute virus of mice (MVM) removal. We used long-term filtrations conducted to a target throughput of 1000 L/m2 with four different monoclonal antibodies on small-scale hollow fiber virus filters with a hydrophilic modified polyvinylidene fluoride membrane. These conditions are certainly challenging for any VF operation and ensuring robust viral clearance under such conditions is critical to the design and implementation of continuous VF. Planova BioEX filters effectively removed MVM at 4 log or greater when run continuously for up to 6 days. Interestingly, pressure increases associated with filter fouling over the duration of long-term filtrations were shown to be reflective of load material variability and could be remediated by implementation of an inline prefilter. Pressure interruptions had minimal impact on overall MVM logarithmic reduction value. Effective virus removal was achieved with pressure increases being largely product-specific, which demonstrates the capability of the virus filter to remove virus independent of pressure increases that are expected to occur with increased protein load.

Antimicrobial activity of silver-copper coating against aerosols containing surrogate respiratory viruses and bacteria

The transmission of bacteria and respiratory viruses through expelled saliva microdroplets and aerosols is a significant concern for healthcare workers, further highlighted during the SARS-CoV-2 pandemic. To address this issue, the development of nanomaterials with antimicrobial properties for use as nanolayers in respiratory protection equipment, such as facemasks or respirators, has emerged as a potential solution. In this study, a silver and copper nanolayer called SakCu® was deposited on one side of a spun-bond polypropylene fabric using the magnetron sputtering technique. The antibacterial and antiviral activity of the AgCu nanolayer was evaluated against droplets falling on the material and aerosols passing through it. The effectiveness of the nanolayer was assessed by measuring viral loads of the enveloped virus SARS-CoV-2 and viability assays using respiratory surrogate viruses, including PaMx54, PaMx60, PaMx61 (ssRNA, Leviviridae), and PhiX174 (ssDNA, Microviridae) as representatives of non-enveloped viruses. Colony forming unit (CFU) determination was employed to evaluate the survival of aerobic and anaerobic bacteria. The results demonstrated a nearly exponential reduction in SARS-CoV-2 viral load, achieving complete viral load reduction after 24 hours of contact incubation with the AgCu nanolayer. Viability assays with the surrogate viruses showed a significant reduction in viral replication between 2–4 hours after contact. The simulated viral filtration system demonstrated inhibition of viral replication ranging from 39% to 64%. The viability assays with PhiX174 exhibited a 2-log reduction in viral replication after 24 hours of contact and a 16.31% inhibition in viral filtration assays. Bacterial growth inhibition varied depending on the species, with reductions ranging from 70% to 92% for aerobic bacteria and over 90% for anaerobic strains. In conclusion, the AgCu nanolayer displayed high bactericidal and antiviral activity in contact and aerosol conditions. Therefore, it holds the potential for incorporation into personal protective equipment to effectively reduce and prevent the transmission of aerosol-borne pathogenic bacteria and respiratory viruses.