Efficient Filtration and Sizing of Viruses with Membrane Filters

Untreated membrane filters retain viruses by adsorption, as well as by physical restriction which occurs when the pore diameter of the filter is smaller than that of the virus particle. As originally recommended by Elford, membranes had to be pretreated with proteinaceous material to preclude virus adsorption. However, coating materials that prevent adsorption of certain viruses do not necessarily prevent adsorption of other viruses. In contrast to proteins, salts enhance virus adsorption. Viruses treated with sodium lauryl sulfate to reduce the surface tension, or purified viruses in distilled water, are not adsorbed to membranes. A procedure is recommended by which viruses may be passed through membranes with a porosity twice the diameter of the virus. Such filtrates, which contain 50 to 100% of the initial virus concentration, should be used for sizing viruses by subsequent filtration through smaller pores. The determination of virus size would then be based on the major population of particles in the virus suspension. In the past, as little as 0.1 to 0.001% of the initial virus population was the basis for size determination, because more than 99.9% of the virus was often lost by adsorption to membranes during the clarifying procedures.     

Membrane Concentration of Infectious Bovine Rhinotracheitis Virus from Water

A membrane adsorption procedure was used to concentrate infectious bovine rhinotracheitis virus from 1-liter quantities of distilled water. Cellulose nitrate membrane filters (0.45-mum pore size) efficiently adsorbed this herpesvirus from water, and virus was recovered from the membrane by elution with 10 ml of fetal calf serum during sonic treatment. The average recovery rate was 70%.     

Membrane Filtration of the Reiter Treponeme

A membrane filtration technique with commercially available membrane filters (Millipore Corp.) was effective for the removal of Reiter treponemes from liquids such as fluorescent-antibody conjugates, to which the organisms are added for adsorption. Reiter treponemes from an 8-day culture were not microscopically detectable in filtrates through membranes with a pore diameter of 0.45 μm, but treponemes were demonstrated in the filtrate by cultural methods. No organisms of the 8-day culture passed through a membrane filter having a pore size of 0.22 μm, as determined by microscopy and culture. Culture data indicated that a filter with a pore size of 0.1 μm was necessary to prevent passage of treponemes from 4-day cultures. It is recommended that a membrane filter with a pore size of 0.22 μm or smaller be used for the removal of Reiter treponemes from suspensions and that the age of the culture be considered in choosing filter pore size.     

Determination of labeled adenine by means of adsorption on cellulose nitrate filters: a sensitive method for estimation of nucleosidase activity.

Cellulose nitrate filters strongly adsorb adenine up to 4.4 nmoles/cm² of the filter with a constant 90% efficiency. Based on this phenomenon a sensitive and fast method of estimation of labeled adenine in amounts over three orders of magnitude was developed. This method has been successfully applied to estimate the activity of minute amounts of a nucleosidase system, catalyzing the decomposition of ATP to adenine. Other purines and pyrimidines are also adsorbed by cellulose nitrate filters in the following order: Adenine > cytosine ? thymine > uracil > guanine. The affinity of corresponding nucleosides as well as nucleotides toward cellulose nitrate filters is greatly reduced. The kinetics of adsorption and desorption of [¹⁴C]adenine was studied with regard to temperature and time of adsorption, additives, source of the filters and their pore size. The mechanism of adsorption is discussed.     

Preparation of stable and solvent-free model membranes.

A method for the preparation of solvent-free, phospholipid-impregnated filters is described. Polycarbonate filters of 13 mm diameter, 5 μm thickness and 0.1 μm pore size were employed, and 20 to 48 nmol phospholipid per filter was incorporated. Passive permeation of polar substances across the filter was determined by a flow-dialysis procedure. The presence of phospholipid led to a decrease in passive permeability by 96 – 99%. Due to their size and stability, and a partially preferential lipid orientation, these phospholipid-impregnated filters may serve as an alternative type of model membrane.     

A simple methodological approach for counting and identifying culturable viruses adsorbed to cellulose nitrate membrane filters

We identified conditions under which Buffalo green monkey cells grew on the surfaces of cellulose nitrate membrane filters in such a way that they covered the entire surface of each filter and penetrated through the pores. When such conditions were used, poliovirus that had previously been adsorbed on the membranes infected the cells and replicated. A plaque assay method and a quantal method (most probable number of cytopathic units) were used to detect and count the viruses adsorbed on the membrane filters. Polioviruses in aqueous suspensions were then concentrated by adsorption to cellulose membrane filters and were subsequently counted without elution, a step which is necessary when the commonly used methods are employed. The pore size of the membrane filter, the sample contents, and the sample volume were optimized for tap water, seawater, and a 0.25 M glycine buffer solution. The numbers of viruses recovered under the optimized conditions were more than 50% greater than the numbers counted by the standard plaque assay. When ceftazidime was added to the assay medium in addition to the antibiotics which are typically used, the method could be used to study natural samples with low and intermediate levels of microbial pollution without decontamination of the samples. This methodological approach also allowed plaque hybridization either directly on cellulose nitrate membranes or on Hybond N+ membranes after the preparations were transferred.     

Concentration of Enteroviruses on Membrane Filters

Enteroviruses can be made to adsorb or to pass through membrane filters by manipulation of the suspending medium. Salts facilitate virus adsorption, but membrane-coating components (MCC) interfere. Because cells release MCC into the culture medium during viral growth, MCC must be removed before virus can be adsorbed to membranes. Adsorbed virus can be eluted with diluents containing MCC (cell extracts or serum) or agents that reduce surface tension (sodium lauryl sulfate). By membrane adsorption and elution, enteroviruses can be readily concentrated and quantitatively recovered from crude virus harvests.     

A Simple Methodological Approach for Counting and Identifying Culturable Viruses Adsorbed to Cellulose Nitrate Membrane Filters

We identified conditions under which Buffalo green monkey cells grew on the surfaces of cellulose nitrate membrane filters in such a way that they covered the entire surface of each filter and penetrated through the pores. When such conditions were used, poliovirus that had previously been adsorbed on the membranes infected the cells and replicated. A plaque assay method and a quantal method (most probable number of cytopathic units) were used to detect and count the viruses adsorbed on the membrane filters. Polioviruses in aqueous suspensions were then concentrated by adsorption to cellulose membrane filters and were subsequently counted without elution, a step which is necessary when the commonly used methods are employed. The pore size of the membrane filter, the sample contents, and the sample volume were optimized for tap water, seawater, and a 0.25 M glycine buffer solution. The numbers of viruses recovered under the optimized conditions were more than 50% greater than the numbers counted by the standard plaque assay. When ceftazidime was added to the assay medium in addition to the antibiotics which are typically used, the method could be used to study natural samples with low and intermediate levels of microbial pollution without decontamination of the samples. This methodological approach also allowed plaque hybridization either directly on cellulose nitrate membranes or on Hybond N+ membranes after the preparations were transferred.     

Concentration of enteroviruses from estuarine water.

Pleated cartridge filters readily adsorb viruses in estuarine water at low pH containing aluminum chloride. Adsorbed viruses are efficiently recovered by treating filters with glycine buffer at high pH. By using these procedures, it was possible to recover approximately 70% of the poliovirus added to 400 liters of estuarine water in 3 liters of filter eluate. Reconcentration of virus in the filter eluate in small volumes that are convenient for viral assays was more difficult. Reconcentration methods described previously for eluates from filters that process tap water or treated wastewater were inadequate when applied to eluates from filters used to process estuarine water containing large amounts of organic compounds. Two methods were found to permit efficient concentration of virus in filter eluates in small volumes. In both methods, virus in 3 liters of filter eluate was adsorbed to aluminum hydroxide flocs and then recovered in approximately 150 ml of buffered fetal calf serum. Additional reductions in volume were achieved by ultrafiltration or hydroextraction. By using these procedures 60 to 80% of the virus in 3 liters of filter eluate could be recovered in a final volume of 10 to 40 ml.     

Concentration of enteroviruses from estuarine water.

Pleated cartridge filters readily adsorb viruses in estuarine water at low pH containing aluminum chloride. Adsorbed viruses are efficiently recovered by treating filters with glycine buffer at high pH. By using these procedures, it was possible to recover approximately 70% of the poliovirus added to 400 liters of estuarine water in 3 liters of filter eluate. Reconcentration of virus in the filter eluate in small volumes that are convenient for viral assays was more difficult. Reconcentration methods described previously for eluates from filters that process tap water or treated wastewater were inadequate when applied to eluates from filters used to process estuarine water containing large amounts of organic compounds. Two methods were found to permit efficient concentration of virus in filter eluates in small volumes. In both methods, virus in 3 liters of filter eluate was adsorbed to aluminum hydroxide flocs and then recovered in approximately 150 ml of buffered fetal calf serum. Additional reductions in volume were achieved by ultrafiltration or hydroextraction. By using these procedures 60 to 80% of the virus in 3 liters of filter eluate could be recovered in a final volume of 10 to 40 ml.     

Influence of salts on virus adsorption to microporous filters

We investigated the direct and indirect effects of mono-, di-, and trivalent salts (NaCl, MgCl(2), and AlCl(3)) on the adsorption of several viruses (MS2, PRD-1, phiX174, and poliovirus 1) to microporous filters at different pH values. The filters studied included Millipore HA (nitrocellulose), Filterite (fiberglass), Whatman (cellulose), and 1MDS (charged-modified fiber) filters. Each of these filters except the Whatman cellulose filters has been used in virus removal and recovery procedures. The direct effects of added salts were considered to be the effects associated with the presence of the soluble salts. The indirect effects of the added salts were considered to be (i) changes in the pH values of solutions and (ii) the formation of insoluble precipitates that could adsorb viruses and be removed by filtration. When direct effects alone were considered, the salts used in this study promoted virus adsorption, interfered with virus adsorption, or had little or no effect on virus adsorption, depending on the filter, the virus, and the salt. Although we were able to confirm previous reports that the addition of aluminum chloride to water enhances virus adsorption to microporous filters, we found that the enhanced adsorption was associated with indirect effects rather than direct effects. The increase in viral adsorption observed when aluminum chloride was added to water was related to the decrease in the pH of the water. Similar results could be obtained by adding HCl. The increased adsorption of viruses in water at pH 7 following addition of aluminum chloride was probably due to flocculation of aluminum, since removal of flocs by filtration greatly reduced the enhancement observed. The only direct effect of aluminum chloride on virus adsorption that we observed was interference with adsorption to microporous filters. Under conditions under which hydrophobic interactions were minimal, aluminum chloride interfered with virus adsorption to Millipore, Filterite, and 1MDS filters. In most cases, less than 10% of the viruses adsorbed to filters in the presence of a multivalent salt and a compound that interfered with hydrophobic interactions (0.1% Tween 80 or 4 M urea).     

Na, k, and nonspecific solute requirements for induction and function of galactose active transport in an antarctic psychrophilic marine bacterium

Strong adherence of bacteria, yeast, erythrocytes, leukocytes, platelets, spores, and polystyrene spheres to membrane filter materials was noted during filtration through membranes with pore size diameters much larger than the particles themselves. Quantitative recovery on the membrane filters of these particles from low-concentration suspensions was achieved during gravity- or vacuum-assisted filtration through membranes with pore diameters as much as 30 times that of the filtered particles. Mechanical sieving was not responsible. The phenomenon was judged to be electrostatic. It could be partially blocked by pretreating the filter with a nonionic surfactant (Tween 20), and elution of adherent particles was achieved with 0.05% Tween 20. Gram-positive cocci were removed from suspension more efficiently than gram-negative rods. The commonly used cellulose membranes adsorbed more bacteria, blood cells, and other particles than did polycarbonate filters. Of lesser adsorptive capacity were vinyl acetate, nylon, acrylic, and Teflon membranes. Backwashing with saline, serum, 6% NaCl, dextran solutions, or phosphate buffers of varying molality and pH removed only a fraction of adherent particles. Tween 20 (0.05%) eluted up to 45% of adherent particles in a single back-filtration. Selected filters quantitatively removed the particles tested, which then could be washed and subjected to reagents for a variety of purposes. It is important to anticipate the removal of particles during membrane filtration, since it is not a simple mechanical event.     

Adherence of bacteria, yeast, blood cells, and latex spheres to large-porosity membrane filters.

Strong adherence of bacteria, yeast, erythrocytes, leukocytes, platelets, spores, and polystyrene spheres to membrane filter materials was noted during filtration through membranes with pore size diameters much larger than the particles themselves. Quantitative recovery on the membrane filters of these particles from low-concentration suspensions was achieved during gravity- or vacuum-assisted filtration through membranes with pore diameters as much as 30 times that of the filtered particles. Mechanical sieving was not responsible. The phenomenon was judged to be electrostatic. It could be partially blocked by pretreating the filter with a nonionic surfactant (Tween 20), and elution of adherent particles was achieved with 0.05% Tween 20. Gram-positive cocci were removed from suspension more efficiently than gram-negative rods. The commonly used cellulose membranes adsorbed more bacteria, blood cells, and other particles than did polycarbonate filters. Of lesser adsorptive capacity were vinyl acetate, nylon, acrylic, and Teflon membranes. Backwashing with saline, serum, 6% NaCl, dextran solutions, or phosphate buffers of varying molality and pH removed only a fraction of adherent particles. Tween 20 (0.05%) eluted up to 45% of adherent particles in a single back-filtration. Selected filters quantitatively removed the particles tested, which then could be washed and subjected to reagents for a variety of purposes. It is important to anticipate the removal of particles during membrane filtration, since it is not a simple mechanical event.     

Integration of Planova filters in manufacturing processes of biologicals improve the virus safety effectively: A review of publicly available data

The capacity to remove viruses by Planova filters produced by Asahi Kasei, primarily by small virus-retentive filters, were compiled from data in peer-reviewed publications and, partly, publicly available data from presentations at conferences (Planova workshops). Data from more than 100 publications and presentations at conferences covering Planova filters were assessed. The data were grouped according to the different virus filters regarding mean pore sizes and viruses of different sizes for plasma and cell culture derived products. Planova 15N and 20N filters removed parvoviruses below the limit of detection of viruses in the filtrate in approx. 50% of all studies and mean LRFs (log reduction factors) for viruses detected in the filtrate were above 4, demonstrating effective parvovirus reduction. Parvovirus removal capacity increased for Planova BioEX filters as well as for 2 Planova 20N in series. Large viruses as retroviruses (e.g., HIV and MuLV), herpesviruses, flaviviruses and togaviruses were removed effectively by Planova 15N, 20N and BioEX filters and also by Planova 35N filters. Flow interruption, transmembrane pressure, volume and protein concentration per filter area had had no substantial impact on virus removal capacity at manufacturing specification. In conclusion, the incorporation of Planova filters in manufacturing processes of biologicals remove, depending on the filter pore size, small and large viruses from the feed stream reliably. This virus reduction step with an orthogonal mechanism integrated in the manufacturing processes of biologicals, based primarily on size exclusion of viruses, improves the virus safety of these biopharmaceutical products considerably.     

Simple method for the concentration of influenza virus from allantoic fluid on microporous filters.

Membrane filter adsorption-elution is an efficient method for concentration and partial purification of several types of viruses from various aqueous solutions. For efficient virus adsorption to negatively charged filters, the sample is adjusted to pH 3.5 and trivalent salts are added before filtration. Since influenza virus is sensitive to extremes in pH, it cannot be concentrated by ordinary filters. Zeta Plus filters, which have a net positive charge of up to 5 or 6, were evaluated for the concentration of influenza virus from infectious allantoic fluids. Influenza virus efficiently adsorbed to Zeta Plus filters at pH 6, and addition of salts was not necessary. Adsorbed virus was eluted in a small volume of 2% bovine serum albumin plus 1 M NaCl at pH 10. By this procedure, viruses in 100 ml of allantoic fluid were concentrated to a final volume of 8 ml, with an average recovery efficiency of 71.0%.     

Poliovirus concentration from tap water with electropositive adsorbent filters.

Simple, reliable, and efficient concentration of poliovirus from tap water was obtained with two types of electropositive filter media, one of which is available in the form of a pleated cartridge filter (Virozorb 1MDS). Virus adsorption from tap water between pH 3.5 and 7.5 was more efficient with electropositive filters than with Filterite filters. Elution of adsorbed viruses was more efficient with beef extract in glycine, pH 9.5, than with glycine-NaOH, pH 11.0. In paired comparative studies, electropositive filters, with adsorption at pH 7.5 and no added polyvalent cation salts, gave less variable virus concentration efficiencies than did Filterite filters with adsorption at pH 3.5 plus added MgCl2. Recovery of poliovirus from 1,000-liter tap water volumes was approximately 30% efficient with both Virozorb 1MDS and Filterite pleated cartridge filters, but the former were much simpler to use. The virus adsorption behavior of these filters appears to be related to their surface charge properties, with more electropositive filters giving more efficient virus adsorption from tap water at higher pH levels.     

Poliovirus concentration from tap water with electropositive adsorbent filters

Simple, reliable, and efficient concentration of poliovirus from tap water was obtained with two types of electropositive filter media, one of which is available in the form of a pleated cartridge filter (Virozorb 1MDS). Virus adsorption from tap water between pH 3.5 and 7.5 was more efficient with electropositive filters than with Filterite filters. Elution of adsorbed viruses was more efficient with beef extract in glycine, pH 9.5, than with glycine-NaOH, pH 11.0. In paired comparative studies, electropositive filters, with adsorption at pH 7.5 and no added polyvalent cation salts, gave less variable virus concentration efficiencies than did Filterite filters with adsorption at pH 3.5 plus added MgCl2. Recovery of poliovirus from 1,000-liter tap water volumes was approximately 30% efficient with both Virozorb 1MDS and Filterite pleated cartridge filters, but the former were much simpler to use. The virus adsorption behavior of these filters appears to be related to their surface charge properties, with more electropositive filters giving more efficient virus adsorption from tap water at higher pH levels.     

Virus filtration of high‐concentration monoclonal antibody solutions

The ability to process high‐concentration monoclonal antibody solutions (> 10 g/L) through small‐pore membranes typically used for virus removal can improve current antibody purification processes by eliminating the need for feed stream dilution, and by reducing filter area, cycle‐time, and costs. In this work, we present the screening of virus filters of varying configurations and materials of construction using MAb solutions with a concentration range of 4–20 g/L. For our MAbs of interest—two different humanized IgG1s—flux decay was not observed up to a filter loading of 200 L/m² with a regenerated cellulose hollow fiber virus removal filter. In contrast, PVDF and PES flat sheet disc membranes were plugged by solutions of these same MAbs with concentrations >4 g/L well before 50 L/m². These results were obtained with purified feed streams containing <2% aggregates, as measured by size exclusion chromatography, where the majority of the aggregate likely was composed of dimers. Differences in filtration flux performance between the two MAbs under similar operating conditions indicate the sensitivity of the system to small differences in protein structure, presumably due to the impact of these differences on nonspecific interactions between the protein and the membrane; these differences cannot be anticipated based on protein pI alone. Virus clearance data with two model viruses (XMuLV and MMV) confirm the ability of hollow fiber membranes with 19 ± 2 nm pore size to achieve at least 3–4 LRV, independent of MAb concentration, over the range examined. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Influence of Salts on Virus Adsorption to Microporous Filters

We investigated the direct and indirect effects of mono-, di-, and trivalent salts (NaCl, MgCl₂, and AlCl₃) on the adsorption of several viruses (MS2, PRD-1, X174, and poliovirus 1) to microporous filters at different pH values. The filters studied included Millipore HA (nitrocellulose), Filterite (fiberglass), Whatman (cellulose), and 1MDS (charged-modified fiber) filters. Each of these filters except the Whatman cellulose filters has been used in virus removal and recovery procedures. The direct effects of added salts were considered to be the effects associated with the presence of the soluble salts. The indirect effects of the added salts were considered to be (i) changes in the pH values of solutions and (ii) the formation of insoluble precipitates that could adsorb viruses and be removed by filtration. When direct effects alone were considered, the salts used in this study promoted virus adsorption, interfered with virus adsorption, or had little or no effect on virus adsorption, depending on the filter, the virus, and the salt. Although we were able to confirm previous reports that the addition of aluminum chloride to water enhances virus adsorption to microporous filters, we found that the enhanced adsorption was associated with indirect effects rather than direct effects. The increase in viral adsorption observed when aluminum chloride was added to water was related to the decrease in the pH of the water. Similar results could be obtained by adding HCl. The increased adsorption of viruses in water at pH 7 following addition of aluminum chloride was probably due to flocculation of aluminum, since removal of flocs by filtration greatly reduced the enhancement observed. The only direct effect of aluminum chloride on virus adsorption that we observed was interference with adsorption to microporous filters. Under conditions under which hydrophobic interactions were minimal, aluminum chloride interfered with virus adsorption to Millipore, Filterite, and 1MDS filters. In most cases, less than 10% of the viruses adsorbed to filters in the presence of a multivalent salt and a compound that interfered with hydrophobic interactions (0.1% Tween 80 or 4 M urea).     

In situ inactivation of animal viruses and a coliphage in nonaerated liquid and semiliquid animal wastes.

The persistence of five animal viruses, representing picorna-, rota-, parvo-, adeno-, and herpesviruses, and the coliphage f2 was determined in the field by exposing the viruses to different animal wastes and by adopting an established filter sandwich technique. This technique allows us to copy the natural state of viruses in the environment, where adsorption onto or incorporation into suspended solids may prolong virus survival. Using filter sandwiches either equipped with porous (15 nm in diameter) or poreless polycarbonate (PC) membranes, it was possible to differentiate between overall virus inactivation and the effect of virucidal agents that act through poreless PC membranes. Depending on ambient temperature, pH, and type of animal waste, values for time, in days, required for a 90% reduction of virus titer varied widely, ranging from less than 1 week for herpesvirus to more than 6 months for rotavirus. Virus inactivation progressed substantially faster in liquid cattle manure, a mixture of urine and water (pH > 8.0), than in semiliquid wastes that consisted of mixtures of feces, urine, water, and bedding materials (pH < 8.0). Hitherto unidentified virucidal agents that permeate poreless PC membranes contributed substantially to the overall inactivation. On the other hand, substances that protect rotavirus and possibly other viruses from inactivation may be present in animal wastes. Together, the study showed that viruses contained in manure may persist for prolonged periods of time if stored under nonaerated conditions. At times of land application, this may lead to environmental contamination with pathogens.