Diffusion and bubble point testing of microporous cartridge filters: preliminary results of production facilities.
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Micropore Filters↗
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Dynamics of protein recovery from process filtration systems using microporous membrane filter cartridges.
The purpose of this report is to address the concern for protein concentration recovery through membrane filter cartridges at the process scale. Filtration systems consisting of pre- and/or final membrane filter cartridges were evaluated for protein concentration recovery under typical manufacturing conditions of continuous flow and high throughput volumes. Results of the study conclusively demonstrate that consideration of the protein adsorptive properties and recovery performance of cartridge filters can provide efficient bio-burden and particulate control without compromising protein yields. Understanding the dynamics of protein recovery through various prefilter and final filter cartridges can play an important role in the proper selection of a filtration system to ensure optimal life and protein yield when filtering dilute protein solutions.
Filtration↗
Diffusion and bubble point testing of microporous cartridge filters: electromechanical methods.
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Diffusion↗
Comparison of two methods for measurement of chemotaxis of neutrophil polymorphonuclear leukocytes in vitro.
Measurement of chemotaxis of neutrophil polymorphonuclear leukocytes (PMNs) is often performed by micropore-filter methods. However, most of these methods seem to involve considerable inter-individual, intra-individual, and day-to-day variations. The aim of this study was to compare two frequently used micropore-filter methods - the Lower Surface Count (LSC) and the Leading Front (LF) methods - in order to find the more reproducible one. Blood samples from the same individual were drawn on two different days. PMN chemotaxis and random migration was tested by a micropore-filter assay. The chemotaxis chambers were incubated for 60, 90, 120, and 150 minutes, respectively. In all, 144 filters were read by both methods. The LSC method showed large variations both among identically treated filters and among readings from the same filter. These variations were as much as 10 times higher than the variations shown by the LF method. Also, the day-to-day variation was higher with the LSC method than with the LF method. Furthermore, data from the LF method were normally distributed, in contrast to data from the LSC method. Thus, the present study suggests the LF method to be superior to the LSC method in terms of reproducibility.
Caseins↗
An ultrasonic analysis of the comparative efficiency of various cardiotomy reservoirs and micropore blood filters.
The ability of 12 commercially available cardiotomy reservoirs to remove bubbles from aspirated blood was investigated by means of a simulated cardiopulmonary bypass circuit and an ultrasonic microbubble detector. Performance varied considerably. The number of gaseous microemboli remaining after passage of blood through the reservoir was reduced by (a) holding the blood in the reservoir, (b) reducing the volume of air mixed with the aspirated blood, and (c) using a reservoir that did not induce turbulence and that contained integral micropore filtration material. Further micropore filtration of the blood after passage through the cardiotomy reservoir was beneficial, and significantly more bubbles were extracted when the microfilter was sited below the reservoir than when it was placed in the arterial line.
Cardiopulmonary Bypass↗
The presence and properties of myocilin in the aqueous humor.
PURPOSE: To determine whether myocilin is present in the aqueous humor (AH) and to examine certain properties of this protein. METHODS: Human AH was obtained at the time of either glaucoma surgery or cataract extraction. Monkey AH was obtained at the time of death, and bovine aqueous was obtained from eyes delivered from an abattoir. Column chromatography was performed on aqueous samples to determine the approximate size of the myocilin present. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis were performed using antibody prepared against a peptide sequence in myocilin. Analysis of the bovine proteins present in AH that were retained by a microporous filter was also performed using western blot analysis. RESULTS: By western blot analysis, myocilin was present in human, monkey, and bovine AH. The apparent molecular size of the myocilin present in the AH were greater than 250,000 Da, when quantified with a gel filtration column. Myocilin appeared to be hydrophobic and was one of the proteins that was retained on microporous filters that were obstructed by AH. CONCLUSIONS: Myocilin is a constituent in the AH. It appears that myocilin is a hydrophobic protein that may exist in an oligomeric state or in association with other proteins. Myocilin is retained by microporous filters and may be involved in the obstruction of these filters that occurs when AH is perfused through them.
Animals↗
[Hemorheological effect of KB-2796, a new Ca2+ antagonist].
The hemorheological effect of KB-2796, 1-[bis(4-fluorophenyl)methyl]-4-(2,3,4,-trimethoxybenzyl)piperazine dihydrochloride, was studied in guinea pigs and rabbits in comparison with those of flunarizine (FNZ) and pentoxifylline (PXF). KB-2796 and FNZ at 10-100 microM dose-dependently prevented crenation of rabbit erythrocytes induced by the Ca2+ ionophore A23187. After incubation of guinea pig whole blood at 37 degrees C, blood micropore-filterability decreased and blood viscosity increased with the progress of erythrocyte crenation. After a 4-hr incubation, KB-2796 inhibited erythrocyte crenation and decreased blood filterability at a concentration of 30 microM, and it increased blood viscosity at 10 microM. Treatment with FNZ (30 microM) and PXF (100 microM) also inhibited erythrocyte crenation and decreased blood filterability. Intravenous administration of KB-2796 at 3 mg/kg significantly inhibited the decrease of blood micropore-filterability after occlusion of the bilateral carotid arteries in rabbits. Although FNZ (3 mg/kg, i.v.) had no effect, PXF (3 mg/kg, i.v.) produced significant inhibition. These results suggest that KB-2796 prevents increase of blood viscosity and decrease of blood filterability by inhibiting the crenation of erythrocytes and suggest that this effect may be useful for the improvement of hemorheology in ischemic disease.
Animals↗
Concentration of viruses from tap water and sewage with a charge-modified filter aid.
Positively charged microporous filters have been shown to have definite advantage over negatively charged filters for concentrating viruses from water as they eliminate the need of water conditioning (acidification and multivalent cation addition) prior to filtration. The present study was designed to evaluate the use of a charge-modified (Zeta plus) filter aid (AMF CUNO, Meriden, CT) for the concentration of viruses (Poliovirus-1, Coxsackievirus B-3 and ECHO virus-7) from large volumes of tap water. Charge-modified filter aid could efficiently adsorb polio, coxsackie and ECHO viruses from water. The adsorbed viruses from both tap water and secondary sewage were most efficiently eluted with 4% beef extract - 0.5 M NaC1, pH 9.5. The efficiency of recovery of poliovirus, coxsackievirus and ECHO virus from 10- to 20-1 vols. of tap water ranged between 32 and 98%, 67 and 100% and 69 and 100%, respectively. This technique appears to have distinct advantages over previous methods as it requires low cost equipment (47 mm polypropylene filter housing) and materials, is simple and easy to handle, can filter large volumes of water (greater than or equal to 20 1) with good recoveries at ambient pH and can be efficiently used to monitor viral water quality.
Adsorption↗
Clearance of murine leukaemia virus from monoclonal antibody solution by a hydrophilic PVDF microporous membrane filter.
Continuous cell lines (used in the manufacture of monoclonal antibodies and other therapeutic proteins) may harbour retroviral particles; nevertheless, they are deemed acceptable if the production system is validated for clearance of these contaminants. As part of a campaign to evaluate and validate appropriate virus removal methodologies for monoclonal immunoglobulin G (IgG) products, studies were undertaken with a hydrophilic polyvinylidene fluoride (PVDF) microporous membrane-the Ultipor(R) VF grade DV50 virus removal membrane. Testing conducted with murine leukaemia virus, a specific model virus used in viral validation studies to document removal of retroviruses, demonstrated a greater that 7 log clearance by the Ultipor(R) VR grade DV50 membrane. Filterability testing established the feasibility of positioning the filter at either of two different stages during manufacture. The results presented in this study demonstrate that membrane filtration through the Ultipor VF grade DV50 virus removal filter can constitute part of the purification regimen to effect removal of endogenous retrovirus-like contaminants from monoclonal IgG products.
Animals↗
Defective mononuclear leukocyte chemotaxis in the Chediak-Higashi syndrome of humans, mink, and cattle.
Chemotaxis of mononuclear leukocytes from humans, mink, and cattle was evaluated in vitro using a morphologic Boyden chamber technique and a new 51-Cr-labeled mononuclear radioassay with a double micropore filter system. Significantly decreased mononuclear leukocyte chemotactic response were noted when human, mink, or cattle Chediak-Higashi cells were tested using autologous serum or endotoxin-activated autolotous serum. A similar Chediak-Higashi mononuclear leukocyte defect was noted in humans when kallikrein or dialyzable transfer factor were used as the chemotactic stimulus. Studies using smaller pore filters in the chemotactic chamber exaggerated the chemotactic defect. Serum from Chediak-Higashi subjects had normal chemotactic activity. Additional studies on the spontaneous (random) locomotion of Chediak-Higashi mononuclear leukocytes revealed normal results when a capillary tube assay system was used, but abnormal results were obtained when a Boyden chamber micropore filter assay was used, demonstrating fundamental differences in these two assays of random locomotion. It is clear from these studies that defective mononuclear leukocyte chemotaxis is another feature of the imparied host defenses in the Chediak-Higashi syndrome that may contribute to the marked susceptibility to pyogenic infections so characteristic of this dease.
Animals↗
Virus retention by a hydrophilic triple-layer PVDF microporous membrane filter.
Retention of bacteriophages (phi 6, PR772, T1, and PP7) and mammalian viruses (poliovirus and influenza A virus) by a hydrophilic triple layer PVDF microporous membrane, the Ultipor VF grade DV50 membrane, was evaluated. Challenges of membrane discs or pleated filter cartridges were performed at concentrations of 10(6)-10(8) PFU/mL in one or more of the following carrier fluids: water, saline, gelatin (0.1%) in phosphate buffer, Dulbecco's Modified Eagle Medium (MEM), and MEM supplemented with 10% fetal bovine serum (MEM + 10). The data demonstrate a minimum log titer reduction (LTR) of 6 for viruses larger than 50 nm irrespective of the carrier fluid. Protein transmission levels of greater than 95% for IgG and albumin were achieved. For integral pleated filter cartridges, correlation between a nondestructive integrity test (using the forward flow integrity test method) and virus retention was demonstrated. The Ultipor VF grade DV50 filter can be applicable in the manufacture of biologicals and biopharmaceuticals, where high protein transmission and consistent viral titer reduction are desired.
Membranes, Artificial↗
Modulation of the random migration of human platelets.
Random migration of human platelets has been recognized as a parameter of platelet function which can be assessed in a reproducible manner by modification of the Boyden micropore filter technique for evaluating this function in other cells (Boyden, S. 1962. J. Exp. Med. 115: 453-466). Because platelets are extremely susceptible to aggregation, the conditions for collecting and isolating platelets and the migration buffer (Ca(++) and Mg(++)-free phosphate buffered saline, pH 6.8, with glucose and gelatin) were selected to minimize such a possibility. The random movement of platelets into the micropore filter was maximal at 30-37 degrees C and was contingent upon the metabolic integrity of the cell; thus, it can be attributed to active spontaneous migration. While the initiating and enhancing effects of epinephrine on the platelet aggregation-release reaction are mediated by an alpha-adrenergic receptor, the inhibition of random migration involved a beta-receptor. Equimolar propranolol but not phentolamine prevented epinephrine inhibition of random migration, and isoproterenol had activity comparable to epinephrine while phenylephrine was inactive. The capacity of the cholinomimetic agent, carbachol, to increase platelet migration is reminiscent of the recent findings in several cell systems in which beta-adrenergic and cholinergic stimuli have opposite effects. The prostaglandins E1 and E2 augmented spontaneous migration in contrast to their well established inhibitory action on platelet aggregation at the concentrations employed. The suppression by indomethacin of prostaglandin enhancement and of spontaneous migration implies a requirement for the prostaglandin biosynthetic pathway during the migration process. Thus, the spontaneous migration of human platelets, an additional parameter of platelet function for in vitro investigations, disclosed not only a beta-adrenergic receptor for epinephrine, but also a capacity for cholinergic augmentation and an apparent requirement for prostaglandin biosynthesis.
Blood Platelets↗
[Locomotion of human circulating lymphocytes methodological aspects "in vitro" (author's transl)].
Locomotor activity of lymphocytes from human peripheral blood towards the chemoattractant casein was examined in vitro in modified Boyden chambers with a single micropore filter (according to the Wilkinson's method), and in Keller's chambers with two micropore filters. Using the single filter technique, this paper reports the precise standard parameters necessary for a reproductive assay, that is a filter of 5 micron pore size (Millipore), an incubation of 5 h with 5 mg/2 ml of alpha-casein, and the counting of lymphocytes at the front of migration within the filter. The distinction between the chemotaxis (directional locomotion) and the chemokinesis (random migration) in the presence of the alpha-casein was confirmed with human circulating lymphocytes. When the results obtained with the double filter technique are compared with that of single filter, Keller's method shows no advantage over the single filter technique modified by Wilkinson. The latter appears adequate to study specific and non-specific drug-induced lymphocyte chemotaxis and chemokinesis with normal or pathological cells in man.
Caseins↗
Quantification of radiolabelled granulocyte migration in vivo.
Quantification of the tissue localisation of granulocytes is difficult, particularly in man. With the aim of facilitating such quantification, a technique is described which combines the micropore filter skin window technique with the infusion of autologous 111In-labelled granulocytes. The radioactivity in the removable micropore filters, placed on small forearm skin window abrasions, was compared with peripheral blood radioactivity following injection of 111In-labelled 'pure' granulocytes in normal subjects. Radioactivity in filters from abrasions which were made 8 h or more before injection of labelled cells followed the same time course as the cell-associated radioactivity in whole blood, but radioactivity in filters from abrasions made nearer to the time of labelled granulocyte injection increased to reach a peak 3-8 h after cell injection, at a time when cell-associated blood radioactivity had fallen to about 50% of its initial value. Exercise appeared to induce a transient decrease in radiolabelled granulocyte migration into the filters. This technique offers a means of studying the kinetics of granulocyte migration in vivo.
Cell Movement↗
[Effects of bFGF and alpha-MSH on adhesion and migration of human melanocytes in vitro].
OBJECTIVE: To observe the effect of basic fibroblast growth factor (bFGF) and alpha-melanocyte stimulating hormone (alpha-MSH) on adhesion and migration of melanocytes in vitro. METHODS: Human melanocytes were obtained from normal human foreskins. Culture dishes covered with fibronectin were used to perform melanocytes adhesion assay, and cell motility was assessed using the Transwell micropore filter method. RESULT: bFGF and alpha-MSH increased melanocytes adhesion on culture dishes covered with fibronectin. bFGF stimulated melanocytes migration through micropore filter while alpha-MSH had no significant effects. CONCLUSION: bFGF and alpha-MSH could promote the adhesion and migration of melanocytes, which suggests that two agents may play a role in the repigmentation of vitiligo.
Cell Adhesion↗
Membrane filters and membrane-filtration processes for health care.
The development of membrane-filtration processes is reviewed, and current types and uses of membrane filtration in health care is discussed. Development of adequate support structures for filters and of disposable filtration devices has facilitated development of filtration processes for pharmaceutical industry, manufacturing in hospital pharmacies, and direct patient care. Hydrophobic filters have also been developed; aqueous solutions cannot wet the pore structures of these filters and therefore cannot pass. Sterility-testing systems have also been developed. There are two types of filters: depth (constructed of compacted fibers) and membrane (which have a homogeneous internal structure). Depth filters retain only a portion of particles in a particular size range and are generally not acceptable for use in health care. Membrane filters retain all particles of a given size. Types of membrane filters are selected for specific uses based on needed flow rates, particulate load, and retention capability. Membrane filters may be validated using bacterial-passage, bubble-point, and diffusion tests. Most membrane filters used in health care are microporous filters that retain particles in the 0.1-10-micron size range. Applications are currently being developed for ultrafilters, which retain both particles and substances with large molecular structures such as proteins, and reverse-osmosis filter membranes, which allow only water or water-miscible solvents of very low molecular weights to pass. Experience in engineering designs, quality assurance, and test procedures has led to the development of many safe, reliable, and effective membrane products for health care.
Micropore Filters↗
Concentration of coliphages from large volumes of water and wastewater.
Membrane filter adsorption-elution technology has been extensively used for the concentration and detection of animal viruses from large volumes of water. This study describes the development of positively charged microporous filters (Zeta Plus) for the concentration of coliphages from large volumes of water and wastewater. Four different coliphages were studied: MS-2, phi X174, T2, and T4. Positively charged microporous filters were found to efficiently adsorb these coliphages from tap water, sewage, and lake water at neutral pH. Adsorbed viruses were eluted with a 1:1 mixture of 8% beef extract and 1 M sodium chloride at pH 9. Using this method, coliphages could be concentrated from 17-liter volumes of tap water with recoveries ranging from 34 to 100%. Coliphages occurring naturally in raw and secondarily treated sewage were recovered with average efficiencies of 56.5 and 55.0%, respectively. This method should be useful in isolation of rare phages, the ecology of phages in natural waters, and the evaluation of water quality.
Coliphages↗
Plasma-induced endothelial activation associated with incident atherosclerosis: prospective results from the Bruneck Study.
BACKGROUND: Whether systemic inflammation is an epiphenomenon of atherosclerosis or whether it is part of the atherosclerosis causal pathway requires further study. DESIGN: As part of a prospective population survey on the course and aetiology of atherosclerosis, we investigated the effects of plasma on the endothelial monolayers inducing activation for leukocyte transmigration. METHODS: An age- and sex-stratified random sample of inhabitants of Bruneck (Italy) with and without atherosclerotic disease aged 50-69 years was selected. Carotid arteries were evaluated by duplex sonography at baseline (1990). Carotid arteries were re-evaluated for the development of new plaques 5 years later (1995). Frozen plasma samples from baseline were available for a random sample of 152 men. Monolayers of endothelial cells cultured in micropore filter insets were pre-treated with plasma, then normal human neutrophils were added to the endothelial cells and subsequent transmigration through the monolayers and micropore filters was measured. RESULTS: The endothelial monolayers were activated for transmigration of leukocytes more potently by plasma from participants with carotid artery plaques than participants without it. Increased endothelial activation with plasma at baseline was associated with the development of new atherosclerotic lesions during a period of 5 years. CONCLUSIONS: Plasma from individuals with prevalent atherosclerosis of the carotid arteries activates the endothelium for leukocyte transmigration, suggesting the presence of systemic pro-inflammatory mediators. In an epidemiological survey, follow-up data on new lesion formation after 5 years indicated that plasma-mediated endothelium activation for interaction with leukocytes precedes the development of atherosclerotic lesions.
Aged↗