Virus Removal Research Articles

Single-Use Technology in Purification of Monoclonal Antibiotics

The advent of Single-Use Technologies (SUT) has revolutionized biopharmaceutical manufacturing, offering a flexible and efficient alternative to traditional methods. This paper explores the diverse applications of SUT, focusing on therapeutic plasma filtration, clarifying processes, virus removal strategies, and chromatography solutions. Highlighting the advantages such as convenience, reduced contamination risk, and streamlined processes, the document delves into the economic and environmental impacts of adopting SUT. Despite challenges in scalability and automation, specific examples like Immusorba PH-350 and Pall Biotech's AllegroTM underscore the significant adsorption efficiency achieved with single-use systems. Anticoagulation strategies tailored for single-use plasma filtration contribute to optimal systemic performance. The dynamic market for disposable devices, featuring prominent manufacturers like Fresenius HemoCare, Kaneka, Medicap Clinic GmbH, Miltenyi Biotec, Toray Medical, and Biotex, reflects the widespread adoption of SUT across various medical applications. The ongoing innovation in overcoming scalability limitations and the commitment to standardization position SUT as a transformative force in modern biopharmaceutical manufacturing. In conclusion, the economic implications and positive environmental impact underscore SUT's potential to shape the future of the industry. Keywords: Single Use Technology, Reduced Contamination, Antibiotics, Biopharmaceutical, Good Manufacturing Practices, Mixing bag, Aseptic Connections, Single use Bioreactors, Disposable Chromatography Columns, Single use Filtration, Running Cost, Labour charges

Impact of membrane surface and module damage on virus removal and integrity in RO membranes

RO membrane modules are very effective in removing viruses and salts, but concerns remain regarding their long-term integrity in full-scale RO spiral wound systems. Membrane defects can arise from delamination, chlorine, or abrasive particle exposure, and module defects can arise from permeate tube, O-ring, and glue line damage. These issues compromise the membrane and module's performance, allowing viruses to pass that are not detected by conductivity monitoring. This paper assesses the impact of damage on virus removal using biological indicators (natural virus markers (NV)) and non-biological surrogates (salt, sulfate, rhodamine WT, pyranine). Three categories of module damage were studied and characterized using different autopsy techniques: membrane module component damage (permeate tube damage, O-ring damage, and membrane delamination), oxidative membrane damage (by hypochlorite exposure dose), and membrane surface damage (by abrasive particle exposure). Module component damage resulted in increased water permeability and decreased natural virus rejection. The conductivity remained similar to that of an intact module except in the case of permeate tube damage. Chlorine exposure (9000 ppm.h) didn't result in detectable NV markers in the permeate, indicating an intact support layer despite active layer damage. Abrasive particle exposure by suspended silicon carbide in the feed resulted in scratches, observed by SEM images, and a decline in the rejection of fluorescent marker, which indicates membrane surface damage. Results demonstrate that NV markers most consistently determine virus removal capacity in modules compromised by component damage. However, for assessing active layer damage, solutes like rhodamine-WT and pyranine prove more effective. This underscores the necessity of employing multiple indicators for a comprehensive evaluation of membrane integrity.

Removal of virus from hands: a study on the role of washing and drying

Proper hand hygiene is highly important to control pathogens' transmission. Although the effect of washing and drying on hands' bacterial contamination has been reported, studies on viruses are scarce. To assess the viral load of artificially contaminated hands after washing and after drying. 30 volunteers filled a questionnaire about hand hygiene and participated in microbial assays, testing five different drying approaches, using the whole hand methodology to quantify viruses on hands. Bacterial assays were also performed for comparison purposes. For both virus and bacteria contaminations, the washing step promoted a significant reduction of the microbial load, while the drying step only promoted slight reductions regardless of the drying method used. Moreover, hand dryers and paper towels didn't induce the re-contamination of washed hands. Hand washing promoted a reduction of the microbial load in hands, while none of the different drying methods significantly reduced hands microbial load.

Development of optimal indoor air disinfection and ventilation protocols for airborne infectious diseases.

Since the COVID-19 pandemic, there has been persistent emphasis on the importance of indoor air disinfection and ventilation in isolation units in the hospital environment. Nevertheless, no optimal and concrete disinfection protocol has been proposed to inactivate the viruses as quickly as possible. In this study, we experimentally evaluated various ventilation and disinfection protocols based on the combination of negative-pressure ventilation, ultraviolet (UV) light illumination, and Hypochlorous acid (HOCl) spray against three active virus species in a 3.5 cubic meters isolation unit. This small-size unit has gained attention during the pandemic due to the high demand for compact mobile laboratory systems capable of rapid disease diagnosis. In accordance with the WHO laboratory biosafety guidance, which states that all enclosed units where diagnostic work is conducted must ensure proper ventilation and disinfection activities, we aim to propose virus removal protocols for units compact enough to be installed within a van or deployed outdoor. The results confirmed the superiority (in terms of virus removal rate and time required) of the virus removal methods in the order of UV light, ventilation, and HOCl spray. Ultimately, we propose two optimal protocols: (i) UV light alone for three minutes, and (ii) UV light with ventilation for three minutes, followed by one-minute ventilation only. The time span of three minutes in the latter protocol is based on the clinical practice such that the medical staffs have a sufficient time to process the samples taken in transition to next patient to care.

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.

Combined performance of upper-room UVGI and ceiling-mounted air cleaners for removing active bioaerosol

Upper-room ultraviolet germicidal irradiation (UR-UVGI) and ceiling-mounted air cleaner (CM-AC) technologies contribute to curbing the spread of infectious diseases such as COVID-19. However, there is a lack of detailed studies on the performance differences between these two control technologies for reducing viral concentrations and infection risk in isolation ward environments with different ventilation modes, especially when used alone versus in combination. Therefore, this study performed numerical simulations under three ventilation modes, namely, downward ventilation (DWV), stratum ventilation (SV), and displacement ventilation (DV), by the Eulerian-Lagrangian method combined with the UV inactivation model. The viral disinfection performance of UR-UVGI and CM-AC was parametrically analyzed. The effectiveness of the combined application of these two control technologies in wards was evaluated. The results showed that expanding the irradiation range was more effective in enhancing the UR-UVGI performance with the same total UV flux. When using UR-UVGI only, DWV disinfected better at the same irradiation flux. In CM-AC cases, the viral aerosol removal efficiency was the greatest in DV. UR-UVGI and CM-AC had advantages in active bioaerosol removal in overall and localized indoor spaces, respectively. The combined application was recommended to maximize the air cleanliness. Under the role of UR-UVGI, a high level of disinfection could be achieved using CM-AC with low ventilation rates. When the CM-AC ventilation rate was 150 m3/h and the UV irradiance was 30 μW/cm2, the viral disinfection of DV exceeded 90%, while that of DWV was also close to 90%. The findings could provide insights to further lower the transmission risk of infectious diseases.

Effect of volumetric concentration factor on virus removal for low-pressure reverse osmosis membrane in drinking water production: A study on different scales

Reverse osmosis membranes are intended to constitute a complete physical barrier against nanometric-sized pathogens such as enteric viruses. Literature describes that low-pressure reverse osmosis achieves high viral removal rates (above 5 log), surpassing those of ultrafiltration (1 to 3 log). However, these studies often used individual viruses and high feed viral concentrations (above 109 virus L-1), greater than typical viral concentrations present in the environment like groundwater, to promote virus detection in the permeate. These high concentrations can promote viral aggregation, potentially affecting the observed retention. This work evaluates the simultaneous elimination of three viruses during the production of drinking water by low-pressure reverse osmosis: two enteric viruses (adenovirus 41 and coxsackievirus-B5) and bacteriophage MS2, a widely used virus surrogate in the literature. The permeates produced by low-pressure reverse osmosis were concentrated to allow virus detection in permeate at lower feed concentrations (106 virus L-1) while staying above the limits of detection and quantification. Experiments were carried out on two pilot plants of different scales (laboratory and semi-industrial) to assess the potential effect of the number of membranes and O-rings on virus retention. The effect of the volume concentration factor on low-pressure reverse osmosis efficiency was evaluated for each scale. Results indicate an average viral reduction of 6 log (up to 7 log), regardless of the size of the virus and/or the scale of LPRO pilot. For the semi-industrial scale, better retention was observed as the volume concentration factor increased. However, viruses were still present in the permeate for each scale (even if close to the detection limit), indicating that retention was not complete. At the same feed viral concentrations, the number of viruses recovered in the semi-industrial scale permeates was higher than in the laboratory scale. A 24-fold greater number of membranes and O-rings used for the semi-industrial scale showed that micro-leaks through O-rings could be responsible for the passage of viruses into the permeate.

Mapping the heterogeneous removal landscape of wastewater virome in effluents of different advanced wastewater treatment systems of swine farm

In advanced wastewater treatment plants on pig farms, meticulous design aims to eliminate intrinsic pollutants such as organic matter, heavy metals, and biological contaminants. In our field survey across Southern China, a notable disparity in wastewater treatment procedures among various farming facilities lies in the utilization of terminal chemical oxidation post-sedimentation tank. However, recent focus in wastewater surveillance has predominantly centered on antibiotic resistance genes, leaving the efficacy of virus removal in different effluent systems largely unexplored. To profile virus composition at the effluent, assess the virus elimination efficiency of chemical oxidation at the effluent end, and the potential environmental driver of virus abundance, we deployed a meta-transcriptomics approach to first determine the total virome in effluent specimens of terminal clean water tank system (CWT) and terminal chemical oxidation system (TCO) in Southern China pig farms, respectively. From these data, 172 viruses were identified, with a median reads per million (RPM) of 27,789 in CWT and 19,982 in TCO. Through the integration of analyses encompassing the co-occurrence patterns within viral communities, the ecology of viral diversity, and a comparative assessment of average variation degrees, we have empirically demonstrated that the procedure of TCO may perturb viral communities and diminish their abundance, particularly impacting RNA viral communities. However, despite the diminished abundance, pathogenic viruses such as PEDV and PRRSV persisted in the effluent following chemical deoxidation at a moderate RPM value, indicating a substantial in situ presence at effluent. Our environmental driver modeling, employing GLM and mantel tests, substantiated the intricate nature of virus community variation within the effluent, influenced heterogeneously by diverse factors. Notably, pond temperature emerged as the foremost determinant, while fishing farming exhibited a positive correlation with virus diversity (p < 0.05). This revelation of the cryptic persistence of virus communities in wastewater effluent expands our understanding of the varied responses of different virus categories to oxidation. Such insights transcend mere virus characterization, offering valuable implications for enhancing biosafety measures in farming practices and informing wastewater-based epidemiological surveillance.

CFD modeling of reactive species air cleaner applications in a classroom

Due to increasing concerns related to airborne virus spread indoors, more reactive species air cleaners are being utilized in classrooms. Reactive species generated by air cleaners decompose airborne pathogens chemically, decreasing the risk of infection. Due to the high reactivity of these oxidants, reactive species may be distributed nonuniformly in indoor environments, as are viral aerosols emitted by infectors. Heterogeneous distributions of reactive species may cause spatially non-uniform removal rates of viral aerosols. However, there is little information regarding spatial distributions of either reactive species or viral aerosols in ventilated classrooms. Thus, the objective of this study was to investigate spatial distributions of reactive species and infectious aerosols and to examine how operating conditions of air cleaners affect viral aerosol removal rates. A CFD model simulated the operation of a reactive species air cleaner generating hydrogen peroxide (H2O2) in a mechanically ventilated 237 m3 classroom with nine occupants. The reactive species air cleaner showed a 3–20 times higher equivalent air change rate to a HEPA filter air cleaner with the same inlet and outlet flows. During the operation of reactive species air cleaners, elevated viral aerosol concentration was confined to regions near infectors. This was due to the high reactivity of reactive species, decreasing the infection probability of receptors from 3.2 % to 0.1 % with a 1-hour exposure time. As the room average concentration of reactive species increased from 15.6 to 50.4 ppb, both below the US Occupational Safety and Health Administration (OSHA) Permissible Exposure Limit (PEL) of 1000 ppb, the room average infection probability decreased from 0.3 % to 0.1 %. Due to the residence times of reactive species, the location of reactive species air cleaners affected the inactivation rate of viral aerosol, resulting in a 24 % variation of concentration difference of infectious aerosol with air cleaner locations.

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.

Reducing moisture content can promote the removal of pathogenic bacteria and viruses from sheep manure compost on the Qinghai-Tibet Plateau

Livestock manure contains a high concentration of pathogenic bacteria, viruses, and other harmful microorganisms. The effective elimination of these pathogens is of great significance to the harmless and resource utilization of livestock and poultry manure. Building upon prior research, this study further explored the removal efficiency of pathogenic bacteria and viruses in sheep manure compost on Qinghai-Tibet Plateau under 45 % (M45) and 60 % (M60) moisture content. The results showed that M45 led to a significant increase in the average composting temperature (P＜0.05), a notable reduction in the levels of Salmonella and Coliforms in the compost products, and facilitated the decline in abundance of various pathogens. As composting progressed, the abundance of viruses declined rapidly. Uroviricota was the most dominant viral phylum, and Siphoviridae, Myoviridae, Herpesviridae, and Podoviridae as the predominant viral families. M45 can significantly reduce the diversity and abundance of compost virus community (P＜0.05). The correlation analysis indicated a negative relationship between pathogens and the average temperature and humus content of the compost. In summary, the reduction of moisture content can enhance the biosafety of sheep manure compost products on the Qinghai-Tibet Plateau. These findings provide theoretical basis and technical support for the harmless and resource utilization of sheep manure waste on the Qinghai-Tibet Plateau.

E3 ubiquitin ligase ANKIB1 attenuates antiviral immune responses by promoting K48-linked polyubiquitination of MAVS

Upon sensing cytosolic viral RNA, retinoic acid-inducible gene-I-like receptors (RLRs) interact with mitochondrial antiviral signaling proteins (MAVSs) to activate IRF3 and nuclear factor κB (NF-κB) signaling, initiating innate immune responses. Thus, RLR activation plays a vital role in the removal of invasive RNA viruses while maintaining immune homeostasis. However, inadequate or excessive activation of immunity can cause harm and can even lead to lethal consequences. In this study, we identify an E3 ligase, ankyrin repeat and IBR domain containing 1 (ANKIB1), which suppresses RLR signaling via MAVS. ANKIB1 binds to MAVS to enhance K48-linked polyubiquitination with K311R, causing proteasomal degradation of MAVS. Deficiency of ANKIB1 significantly increases the RLR-mediated production of type I interferon (IFN) along with pro-inflammatory factors. Consequently, ANKIB1 deficiency remarkably increases antiviral immunity and decreases viral replication invivo. Therefore, we reveal that ANKIB1 restricts RLR-induced innate immune activation, indicating its potential role as a therapeutic target for viral infections.

Magnetic biochar conjugated quaternary ammonium salt-constructed hemoperfusion adsorbent for viral removal with rapid and high-performance

Due to variations in virus structures and their high variability, treating viral infections remains challenging. Moreover, developing vaccines and medications for newly emerged viruses demands a significant time investment. Efficient methods are required to prevent dangerous infections caused by unknown pathogens. Hemoperfusion, with its low cost, can effectively remove toxins from the blood quickly, thus achieving therapeutic effects. The core of the hemoperfusion technology lies in the adsorbent. Therefore, the development of a viral adsorbent aiming to combat viruses through hemoperfusion was envisioned, following general principles to effectively save lives and buy time for the research and development of vaccines and medications. Towards this goal, a facile, green, and low-priced magnetic biochar/quaternary ammonium salt (Fe3O4/C-2) was constructed using the hydrothermal carbonization (HTC) method. HIV lentiviruses were used as a model pathogen to quantify the adsorption capacity and elucidate the adsorption mechanism. Benefiting from a macroporous structure and electrostatic interactions, fabricated Fe3O4/C-2 has a high adsorption capacity (965 ng g−1) and strong suppression of infectious activity (99.99%). Moreover, a low hemolysis ratio (<2%), high cell viability (>100%), high activated partial thromboplastin time (APTT) (67.29 s), and low protein adsorption (<5%) indicate outstanding biocompatibility and good anticoagulant activity. Therefore, Fe3O4/C-2 is expected to serve as a simple and efficient pathogen sorbent for viral diseases treatment.

3D‐Printed Highly Porous Functional Materials for the Efficient Removal of Adenovirus

AbstractHierarchical porous acrylate‐based materials are highly interesting as 3D filter materials, such as for virus removal from suspensions. Here, the synthesis of highly porous monolithic 3D materials by polymerization‐induced phase separation in liquid crystal display (LCD) based 3D printing is presented for the efficient removal of human adenovirus type 5. The hierarchical porosity can be tuned via the variation of the photocurable resin composition (i.e., inherent porosity) and the computer‐aided design (i.e., “printed” porosity; microchannels). 3D polymer structures with highly intricate geometries and structural features ranging from ≈20 nm up to cm can be achieved, which can be used for effective virus removal in a laboratory‐scale flow‐through approach. Combined focused ion beam/scanning electron microscopy tomography and mercury porosimetry provide detailed information on the inherent pore size, pore size distribution, and pore interconnectivity, which is key for the performance of such functional 3D materials. Polymers with a theoretical void volume of 75% show virus capture with a removal efficiency of ≈70% of the adenovirus. Polymers with the same theoretical void volume and macroscopic design but a more hydrophobic nature captured only ≈33%. An optimized adenovirus retention of 98% is achieved by adjusting the microchannels of the tunable inserts.

Comparative Removal of Poliovirus, Rotavirus SA11 and MS2 Coliphage by Point-of-Use Devices used to Treat Drinking Water and Water Disinfectants: A Review.

Test protocols have been developed to test water treatment devices/systems for use for treating drinking water that are used at the individual and home level to ensure the removal of waterborne viruses. Current test procedures call for the use of poliovirus type 1 and/or rotavirus SA11. Recently we suggested that selected coliphages could be used as surrogates for poliovirus for testing of point-of-use (POU) water treatment devices, however, rotavirus was not used in those studies. The purpose of this review was to compare studies of POU devices which were tested with poliovirus type 1, simian rotavirus SA11 and coliphage MS2 to determine if the behavior of rotavirus SA11 was significantly different. In addition, an attempt was made to compare the relative resistance of these viruses by various disinfectants used to treat drinking water. In all cases SA11 was removed to an equal or greater degree than poliovirus. SA11 was found to be less resistant to halogens, although one study found it to be more resistance to chloramines than poliovirus and MS2. Based on this review, use of coliphages for testing POU devices appear justified. Additionally, data on chloramines for these viruses would be useful to determine if rotavirus is more resistant than poliovirus and MS2.

Mechanistic modeling of minute virus of mice surrogate removal by anion exchange chromatography in micro scale

Biopharmaceutical products are often produced in Chinese hamster ovary (CHO) cell cultures that are vulnerable to virus infections. Therefore, it is a regulatory requirement that downstream purification steps for biopharmaceuticals can remove viruses from feedstocks. Anion exchange chromatography (AEX) is one of the downstream unit operations that is most frequently used for this purpose and claimed for its capability to remove viruses. However, the impact of various process parameters on virus removal by AEX is still not fully understood. Mechanistic modeling could be a promising way to approach this gap, as these models require comparatively few experiments for calibration. This makes them a valuable tool to improve understanding of viral clearance, especially since virus spiking studies are costly and time consuming.In this study, we present how the virus clearance of a MVM mock virus particle by Q Sepharose FF resin can be described by mechanistic modeling. A lumped kinetic model was combined with a steric mass action model and calibrated at micro scale using three linear gradient experiments and an incremental step elution gradient. The model was subsequently verified for its capability to predict the effect of different sodium chloride concentrations, as well as residence times, on virus clearance and was in good agreement with the LRVs of the verification runs. Overall, models like this could enhance the mechanistic understanding of viral clearance mechanisms and thereby contribute to the development of more efficient and safer biopharmaceutical downstream processes.

Triple filtration robot (T-robot) based on functionally graded multilayer antiviral environment-friendly (F-MAX) system for pathogen purification in confined space

The improved abstract which especially take the suggestions above can be seen as follow:This study addresses the challenge of pathogen regulation in confined spaces by introducing the T-robot, an innovative air filtration robot featuring the F-MAX multilayer composite plate. Designed to capture a wide range of pollutants, including harmful viruses and bacteria, the T-robot significantly enhances air quality. The experimental setup used magnesium phosphate cement, electrostatically charged melt-blown fabric, and eco-friendly materials such as lithium brine by-product magnesia. Key results include a virus removal rate of 99.99% and an antibacterial rate of 98%.The F-MAX system combines multiple layers, each targeting specific particles, with features like the self-healing Desert Rose (DR) coating and high-speed air circulation. The T-robot's high filtration efficiency and sustainable design make it superior to traditional methods, suitable for both commercial and residential use. Its durability and advanced filtration capabilities help reduce airborne contaminants, creating healthier living spaces and demonstrating a commitment to a sustainable future.

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.

Improving Downstream Process Related Manufacturability Based on Protein Engineering—A Feasibility Study

ABSTRACTWhile bioactivity and a favorable safety profile for biotherapeutics is of utmost importance, manufacturability is also worth of consideration to ease the manufacturing process. Manufacturability in the scientific literature is mostly related to stability of formulated drug substances, with limited focus on downstream process‐related manufacturability, that is, how easily can a protein be purified. Process‐related impurities or biological impurities like viruses and host cell proteins (HCP) are present in the harvest which have mostly acid isoelectric points and need to be removed to ensure patient safety. Therefore, during molecule design, the surface charge of the target molecule should preferably differ sufficiently from the surface charge of the impurities to enable an efficient purification strategy. In this feasibility study, we evaluated the possibility of improving manufacturability by adapting the surface charge of the target protein. We generated several variants of a GLP1‐receptor‐agonist‐Fc‐domain‐FGF21‐fusion protein and demonstrated proof of concept exemplarily for an anion exchange chromatography step which then can be operated at high pH values with maximal product recovery allowing removal of HCP and viruses. Altering the surface charge distribution of biotherapeutic proteins can thus be useful allowing for an efficient manufacturing process for removing HCP and viruses, thereby reducing manufacturing costs.