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1.
Isolation and characterization of putative trefoil peptide receptors 总被引:13,自引:0,他引:13
Mammalian trefoil factors (TFFs) constitute a group of three peptides (TFF1, TFF2 and TFF3) widely distributed in the gastrointestinal tract. Although a mucosal protection/healing effect of these peptides is well documented the mechanism of action is still unknown. A mucosal membrane extract was prepared from porcine stomach scrapings and incubated with a gel containing immobilized porcine TFF2. The affinity gel material was specifically eluted with a neutral buffer containing a high concentration of the ligand (porcine TFF2). A subsequent SDS–gel electrophoresis showed one protein with a MW of approximately 220 kDa and three proteins with MW around 140 kDa. The proteins were analyzed by trypsin digestion followed by mass spectrometric sequencing of tryptic fragments. In this way a 140-kDa β subunit of fibronectin receptor and a 224-kDa CRP-Ductin gene product were identified. The CRP-Ductin gene product (also named MUCLIN), which is expressed in the intestinal crypts, is characterized by being a membrane protein with a short cytoplasmic region, a transmembrane domain and a large extracellular region. This protein thus fulfils some of the criteria for being a TFF receptor or a TFF binding protein. 相似文献
2.
The Predictive Aggregate Transport Model for microfiltration is used in combination with optimum fluid mechanics and electrostatics to maximize recovery of a heterologous immunoglobulin (IgG) from transgenic goat milk. The optimization algorithm involved varying pH (6.8-9), transmembrane pressure (2-4.5 psi), milk feed concentration (1-2X), membrane module type (linear vs. helical design), and axial velocity (Reynolds number: 830-1170). Operation in the pressure-dependent regime at low uniform transmembrane pressures (approximately 2 psi) using permeate circulation in co-flow, at the pI of the protein (9 in this case) was used to increase IgG recovery from less than 1% to over 95%. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and attenuated total reflection Fourier transform infrared spectroscopy of the microfiltration permeate samples confirmed that all the fat globules and most of the casein micelles were retained in the MF membrane whereas a large amount of the target IgG was transported through the membrane. Transmembrane pressure and hence permeation flux was kept low (approximately 15 lmh) to maximize IgG membrane transport and thus recovery, due to a sparse deposit on the membrane which facilitated high solute transport. Next, an analytical method was used to optimize the diafiltration process using the aggregate transport model, experimental target protein sieving coefficients and permeation flux (Baruah and Belfort, 2003). The methodology reported here should be generalizable to the recovery of target proteins found in other complex suspensions of biological origin using the microfiltration process. 相似文献
3.
With the goal of recovering heterologous immunoglobulin (IgG), which comprises 10-15% of the total proteins, from transgenic goat milk at 80% yield and 80% purity, we have developed and tested a two-step membrane isolation and purification process. In the first step, reported earlier by Baruah and Belfort, microfiltration was used to fractionate the milk proteins and recover > 90% of the original IgG at a purity of about 15-20% in the permeate stream. Here, we focus on ultrafiltration (UF) to increase the purity of the target protein to 80%, while maintaining a relatively high IgG yield (80%). Tangential flow UF experiments in diafiltration mode were conducted with 100 kDa cellulosic membranes to evaluate the optimal pH, ionic strength, and uniform transmembrane pressure (TMP). The TMP was kept uniform by permeate circulation in co-flow mode. The traditional approach of conducting the UF process close to the pI of the predominant whey proteins (15-40 kDa, pI 5.2), to transmit these proteins while retaining heterologous IgG (155 kDa), could not be applied here because of precipitation of residual casein at pH values lower than 8.5. Instead, the packing characteristics of the cake layer on the membrane wall, as elucidated in the Aggregate Transport Model presented by Baruah et al. was utilized to achieve a selectivity of > 15, which was sufficient to meet the stated goals of purity and yield for this difficult separation. This combined process is expected to reduce the load on subsequent purification and polishing steps for eventual therapeutic use. 相似文献
4.
Experiments were performed on a cellulose acetate ultrafiltration membrane (HF-200, ABCOR Inc., Cambridge, Mass.) to test its efficacy in concentrating and purifying a crude enzyme (trypsin) preparation. Studies were also made to determine the influence of inorganic salts, pressure, and temperature on the rate of ultrafiltration for this membrane. The results showed reductions in the rates will be encountered due to the presence of inorganic salts. However, the reduced rates were still sufficiently high to make this method extremely attractive. Operating at filtration pressures above 75 psi at, 20 to 30°C for this membrane does not show any beneficial effect in terms of ultrafiltration rates. However, at 10°C there were continual increases in the filtration rates up to 100 psi. Concentration and purification studies with trypsin yielded a concentration factor of 8.35 and a purification factor 2.35. It was shown concretely that the purification of the enzyme was due to the passage of low molecular weight proteins (below 20,000) through the membrane. Enzyme activity slightly greater than 90% was obtained: 70% was found in the concentrate and 20% in the filtrate. It is concluded that membrane ultrafiltration is an ideal simple, rapid, and economical method for the recovery of biological active substances. 相似文献
5.
Removal of RNA impurities by tangential flow filtration in an RNase-free plasmid DNA purification process 总被引:9,自引:0,他引:9
Addition of animal-derived ribonuclease A to degrade RNA impurities is not recommended in the manufacture of pharmaceutical-grade plasmid DNA. Tangential flow filtration (TFF) takes advantage of the significant size difference between RNA and plasmid DNA to remove RNA in the permeate while plasmid remains in the retentate, in an RNase-free plasmid purification process. Operating conditions including transmembrane pressure, membrane pore size, conductivity of the diafiltration buffer, and plasmid load on the membrane were investigated to maximize RNA clearance. Although direct TFF of clarified lysate removed substantial amounts of RNA, the RNA levels left in the retentate were still significant. Calcium chloride is a potent precipitant of high-molecular-weight RNA. The addition of calcium chloride to the clarified lysate combined with the clearance of low-molecular-weight RNA by TFF resulted in complete RNA removal and high plasmid recovery. 相似文献
6.
Recovery of protein from poultry processing wastewater using membrane ultrafiltration 总被引:1,自引:0,他引:1
The feasibility of using ultrafiltration (UF) to recover protein from poultry processing wastewater (PPW) after primary treatment was investigated. By using polysulfone membrane with 30,000 molecular-weight-cut-off, almost all crude proteins in PPW were retained, subsequently reducing the chemical oxygen demand (COD) in the effluent to less than 200 mg L(-1). Similar to the processing of proteinaceous materials, the average fluxes reached only 100 Lm(-2) h(-1). By identifying the optimal values of key operation parameters, including pH, volumetric flow rate, and transmembrane pressure (6.74, 683 mL min(-1) and 14 psi, respectively) using response surface methodology (RSM), the flux was improved to higher than 200 Lm(-2) h(-1). Although severe membrane fouling was still inevitable after processing, flushing the membrane with a cleaning reagent was found capable of effectively restoring membrane performance. 相似文献
7.
High performance tangential flow filtration 总被引:2,自引:0,他引:2
van Reis R Gadam S Frautschy LN Orlando S Goodrich EM Saksena S Kuriyel R Simpson CM Pearl S Zydney AL 《Biotechnology and bioengineering》1997,56(1):71-82
Conventional tangential flow filtration (TFF) has traditionally been limited to separation of solutes that differ by about ten-fold in size. Wide pore-size distributions, membrane fouling, and concentration polarization phenomena have commonly been cited as reasons for this limitation. The use of TFF in the biotechnology industry has therefore been restricted to cell-protein, virus-protein, and protein-buffer separations. A multi-disciplinary team with industrial and academic members was formed to overcome these limitations and enable protein-protein separations using High Performance TFF (HPTFF) systems. Pore-size distributions have been improved with the development of new membrane formulation and casting techniques. Membrane fouling has been controlled by operating in the transmembrane pressure-dependent regime of the filtrate flux curve and by carefully controlling fluid dynamic start-up conditions. Concentration polarization was exploited to enhance, rather than limit, the resolution of solutes. Concentration polarization has also been controlled by operating a co-current filtrate stream that maintains transmembrane pressure constant along the length of the TFF module. High yields and purification factors were obtained even with small differences in protein sieving. IgG-BSA and BSA monomer-oligomer mixtures have successfully been separated with these systems. HPTFF technology provides a competitive purification tool to complement chromatographic processing of proteins. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 71-82, 1997. 相似文献
8.
Characterizing the impact of pressure on virus filtration processes and establishing design spaces to ensure effective parvovirus removal
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Daniel Strauss Joshua Goldstein Tomoko Hongo‐Hirasaki Yoshiro Yokoyama Naokatsu Hirotomi Tomoyuki Miyabayashi Dominick Vacante 《Biotechnology progress》2017,33(5):1294-1302
Virus filtration provides robust removal of potential viral contaminants and is a critical step during the manufacture of biotherapeutic products. However, recent studies have shown that small virus removal can be impacted by low operating pressure and depressurization. To better understand the impact of these conditions and to define robust virus filtration design spaces, we conducted multivariate analyses to evaluate parvovirus removal over wide ranges of operating pressure, solution pH, and conductivity for three mAb products on Planova? BioEX and 20N filters. Pressure ranges from 0.69 to 3.43 bar (10.0–49.7 psi) for Planova BioEX filters and from 0.50 to 1.10 bar (7.3 to 16.0 psi) for Planova 20N filters were identified as ranges over which effective removal of parvovirus is achieved for different products over wide ranges of pH and conductivity. Viral clearance at operating pressure below the robust pressure range suggests that effective parvovirus removal can be achieved at low pressure but that Minute virus of mice (MVM) logarithmic reduction value (LRV) results may be impacted by product and solution conditions. These results establish robust design spaces for Planova BioEX and 20N filters where high parvovirus clearance can be expected for most antibody products and provide further understanding of viral clearance mechanisms. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1294–1302, 2017 相似文献
9.
Purification of lysozyme using ultrafiltration 总被引:2,自引:0,他引:2
This article examines the separation of lysozyme from chicken egg white by ultrafiltration with 25 kDa and 50 kDa MWCO polysulfone membranes. The effects of pH, system hydrodynamics, feed concentration, and transmembrane pressure on permeate flux, lysozyme transmission, purification factor, and productivity have been discussed. With both types of membranes, higher permeate flux and lysozyme transmission were observed at higher pH. Higher lysozyme purity was generally obtained with the 25 kDa MWCO membrane. Purity of lysozyme decreased when the feed concentration was increased. With the 50 kDa MWCO membrane permeate flux, productivity and the purity of lysozyme were found to increase with increase in transmembrane pressure. The possibility of using a two-step ultrafiltration process for achieving high productivity along with high purity of lysozyme was also investigated. 相似文献
10.
Separation of protease, trypsin and chymotrypsin from yellowfin tuna spleen extract by ultrafiltration (UF) using regenerated cellulose membranes with molecular weight cut off (MWCO) 30 and 100 kDa was studied. The 100 kDa membrane had a higher transmission of enzymes than that of the 30 kDa membrane. The enzyme transmission varied from 0.01 to 0.18 and from 0.6 to 0.8 for the 30 kDa membrane and 100 kDa membrane, respectively. The protein transmission was about 0.8 for both membranes. Increasing cross-flow rate and transmembrane pressure (TMP) increased permeate flux. The limiting fluxes at cross-flow rate 120, 240 and 360 L/h for the 30 kDa membrane were 17.3, 43.9 and 54.7 L/m2h, respectively and the limiting fluxes at the same flow rate for 100 kDa membrane were 34.1, 51.1 and 68.4 L/m2h, respectively. The separation of these proteases was achieved using the 30 kDa membrane. The purities of proteases were increased more than ten times at TMP 1.5 bar and cross-flow rate 360 L/h by diafiltration using 30 kDa membrane. 相似文献
11.
Evaluation of membranes for use in on-line cell separation during mammalian cell perfusion processes
In this study two microporous hollow fibre membranes were evaluated for their use as cell retention device in continuous perfusion systems. A chemically modified permanent hydrophillic PTFE membrane and a hydrophilized PP membrane were tested. To investigate the filtration characteristics under process conditions each membrane was tested during a long term perfusion cultivation of a hybridoma cell line. In both cultivations the conditions influencing membrane filtration (e.g. transmembrane flux) were kept constant. Filtration behaviour was investigated by monitoring transmembrane pressure and protein permeability. Transmembrane pressure was measured on-line with an autoclavable piezo-resistive pressure sensor. Protein permeability was determined by quantitative evaluation of unreduced, Coomassie stained SDS-PAGE. The membrane fouling process influences the filtration characteristics of both membranes in a different way. After fermentation the PP membrane was blocked by a thick gel layer located in the big outer pores of the asymmetric membrane structure. The hydraulic resistance was higher but the protein permeability was slightly better than of the PTFE membrane. For this reason the PP membrane should be preferred. On the other hand, transmembrane pressure decreases slower when the PTFE membrane is used, which favours this membrane for long term cultivations, especially when low molecular weight proteins (<30 KD) are produced.Abbreviations PP
Polypropylene
- PTFE
Polytetrafluoroethylene 相似文献
12.
Hiroyuki Satofuka Yoko Okabe Yuki Takano Takahiko Utsugi Masaya Ohtsu Yasufumi Murakami 《Biochemical and biophysical research communications》2014
A novel method using metastatic breast cancer cell lines was established for producing monoclonal antibodies (mAbs) against multi-span membrane proteins. Grafting of metastatic cells (MCF7-14) into the mammary gland of BALB/cJ/nu/nu mice induced splenic hypertrophy (1.6–3.0 × 108 cells/spleen [n = 6]). More than half of the mAbs against MCF7-14 cells reacted with the cell membrane. Inducing production of antibodies against the extracellular domain of multi-pass membrane proteins is difficult. Because the protein structure becomes more complex as the number of transmembrane domains increases, preparing antigens for immunization in which the original structure is maintained is challenging. Using highly metastatic MDA-MB231 cells as the host cell line, we produced mAbs against a 12 transmembrane protein, solute carrier family 6 member 6 (SLC6A6), as a model antigen. When SLC6A6-overexpressing MDA-MB231 cells were grafted into nude mice, the number of splenocytes increased to 2.7–11.4 × 108 cells/spleen (n = 10). Seven mAb-producing clones that not only recognized the extracellular domain of SLC6A6 but also were of the IgG subclass were obtained. Immunocytochemistry and flow cytometry analyses revealed that these mAbs recognized the native form of the extracellular domain of SLC6A6 on the cell surface. Our novel immunization method involving highly metastatic cells could be used to develop therapeutic mAbs against other multi-pass membrane proteins. 相似文献
13.
Monoclonal antibodies (mAbs) against Toxoplasma gondii, Tg378 and Tg556 clones, are specifically observed to localize to the dense granules of tachyzoites by immunofluorescence microscopy. mAb Tg556 is directed against GRA3, a previously described 30kDa dense granular protein. mAb Tg378 is directed against a novel 36kDa dense granular protein, which we refer to as GRA10. These are major proteins in the excretory/secretory proteins from T. gondii before the parasite's entry into host cells, and they are released into the parasitophorous vacuole (PV) during or shortly after invasion to be associated with the PV membrane. GRA10 binds to the membrane of the host cells regardless of its anchorage-dependence or -independence. The cDNA sequence encoding GRA10 was determined by screening a T. gondii cDNA expression library with mAb Tg378. The deduced amino acid sequence of GRA10 consists of a polypeptide of 364 amino acids, and it has no significant homology to any other known proteins. The sequence contains amino terminal signal peptides and two potential transmembrane domains in the middle of sequence that are not near the carboxy terminus. GRA10 has a RGD motif between the two potential transmembrane domains. 相似文献
14.
Saichit Khummuang Waraporn Phanphrom Witida Laopajon Watchara Kasinrerk Ponlatham Chaiyarit Supansa Pata 《Biological procedures online》2017,19(1):14
Background
Human trefoil factor (TFF) peptides consist of three members: TFF1, TFF2 and TFF3. TFF3 is the most abundant TFF peptide in saliva. TFF3 homodimer was suggested to be involved in apoptosis inhibition and malignancy. Determination of TFF3 homodimer expression profiles in saliva may lead to new information about oral biology and diseases. The objective of this study was to generate monoclonal antibodies (mAbs) against TFF3 and apply the produced mAbs for the establishment of ELISA for quantification of dimeric TFF3 in saliva.Results
With our modified hybridoma technique, three hybridoma clones producing anti-TFF3 mAbs having IgG isotype were generated. The mAbs were specific for TFF3 with no cross-reactivity to other TFFs. Using the generated mAbs, a modified-sandwich ELISA with high sensitivity for the quantification of dimeric TFF3 in saliva was developed. Using this ELISA, the amount of dimeric TFF3 in saliva could be measured.Conclusions
A modified-sandwich ELISA for the quantification of TFF3 dimeric form was established. The established ELISA will be a valuable tool for facilitating the investigation of the physiological roles and the diagnostic values of TFF3 in oral diseases. The concept of this modified-sandwich ELISA may be applied for the determination of other homodimeric peptides of interest.15.
Interaction between TFF1, a gastric tumor suppressor trefoil protein, and TFIZ1, a brichos domain-containing protein with homology to SP-C 总被引:3,自引:0,他引:3
TFF1 is a gastric tumor suppressor that protects gastric epithelial cells from damage but can promote invasive properties of tumor cells. Antibodies were raised against correctly folded TFF1 protein. These showed that the 6.67 kDa secreted trefoil protein is present as an approximately 25 kDa complex in normal human gastric mucosa. The TFF1 complex was immunopurified from human gastric mucosa and shown to comprise two proteins joined by a disulfide bond. Both were identified by amino-terminal sequencing and MALDI TOF mass spectrometry. The TFF1 protein partner is a previously unknown protein that we have called TFIZ1 for trefoil factor interactions(z) 1. TFIZ1 is expressed and secreted in normal gastric mucosa. TFIZ1 mRNA was cloned from gastric mucosa and sequenced. TFIZ1 is an 18.31 kDa protein and contains an approximately 100 amino acid brichos domain and homology with smart00019.10, SF_P. This is the first demonstration that a member of the trefoil factor family of proteins is bound covalently to a brichos domain-containing protein. The apparent molecular mass of the TFF1:TFIZ1 heterodimer is remarkably close to the theoretical molecular mass of 24.98 kDa. In conclusion, the heterodimer comprises one molecule each of TFF1 and TFIZ1, and the disulfide bond between TFF1 and TFIZ1 is the most important factor stabilizing the heterodimer. 相似文献
16.
Schlaeppi JM Henke M Mahnke M Hartmann S Schmitz R Pouliquen Y Kerins B Weber E Kolbinger F Kocher HP 《Protein expression and purification》2006,50(2):185-195
The efficient preparation of recombinant proteins at the lab-scale level is essential for drug discovery, in particular for structural biology, protein interaction studies and drug screening. The Baculovirus insect-cell expression system is one of the most widely applied and highly successful systems for production of recombinant functional proteins. However, the use of eukaryotic cells as host organisms and the multi-step protocol required for the generation of sufficient virus and protein has limited its adaptation to industrialized high-throughput operation. We have developed an integrated large-scale process for continuous and partially automated protein production in the Baculovirus system. The instrumental platform includes parallel insect-cell fermentation in 10L BioWave reactors, cell harvesting and lysis by tangential flow filtration (TFF) using two custom-made filtration units and automated purification by multi-dimensional chromatography. The use of disposable materials (bags, filters and tubing), automated cleaning cycles and column regeneration, prevent any cross-contamination between runs. The preparation of the clear cell lysate by sequential TFF takes less than 2 h and represents considerable time saving compared to standard cell harvesting and lysis by sonication and ultra-centrifugation. The process has been validated with 41 His-tagged proteins with molecular weights ranging from 20 to 160 kDa. These proteins represented several families, and included 23 members of the deubiquitinating enzyme (DUB) family. Each down-stream unit can process four proteins in less than 24 h with final yields between 1 and 100 mg, and purities between 50 and 95%. 相似文献
17.
Marie‐Françoise Clincke Carin Mölleryd Ye Zhang Eva Lindskog Kieron Walsh Véronique Chotteau 《Biotechnology progress》2013,29(3):754-767
High cell density perfusion process of antibody producing CHO cells was developed in disposable WAVE Bioreactor? using external hollow fiber filter as cell separation device. Both “classical” tangential flow filtration (TFF) and alternating tangential flow system (ATF) equipment were used and compared. Consistency of both TFF‐ and ATF‐based cultures was shown at 20–35 × 106 cells/mL density stabilized by cell bleeds. To minimize the nutrients deprivation and by‐product accumulation, a perfusion rate correlated to the cell density was applied. The cells were maintained by cell bleeds at density 0.9–1.3 × 108 cells/mL in growing state and at high viability for more than 2 weeks. Finally, with the present settings, maximal cell densities of 2.14 × 108 cells/mL, achieved for the first time in a wave‐induced bioreactor, and 1.32 × 108 cells/mL were reached using TFF and ATF systems, respectively. Using TFF, the cell density was limited by the membrane capacity for the encountered high viscosity and by the pCO2 level. Using ATF, the cell density was limited by the vacuum capacity failing to pull the highly viscous fluid. Thus, the TFF system allowed reaching higher cell densities. The TFF inlet pressure was highly correlated to the viscosity leading to the development of a model of this pressure, which is a useful tool for hollow fiber design of TFF and ATF. At very high cell density, the viscosity introduced physical limitations. This led us to recommend cell densities under 1.46 × 108 cell/mL based on the analysis of the theoretical distance between the cells for the present cell line. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:754–767, 2013 相似文献
18.
A shortcut method for evaluation of protein deposition onto the membrane surface in crossflow ultrafiltration
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In this study, a procedure for quantifying the surface deposition of proteins in crossflow ultrafiltration has been developed. The procedure consists of determining the protein adsorption behavior onto the membrane surface from a few dynamic measurements performed in a nonfiltration and a filtration mode, and evaluating the concentration polarization (CP) layer thickness based on the adsorption data. To predict the interdependence between the protein adsorption and CP, a simplified mathematical model has been formulated. The model was used to assess the protein adsorption and thus yield reduction in the ultrafiltration process at different protein concentration in the solution. As a case study, ultrafiltration of aqueous solutions of BSA and lysozyme (LYZ) was examined on a polyethersulfone membrane with the molecular weight cutoff of 10 or 100 kDa. The protein concentration in the solutions varied within a relatively low concentration range, i.e. below 10 mg mL?1, characteristic for solvent exchange between sequential operations of protein purification by chromatography and extraction. Both proteins markedly differed in the mechanism of surface deposition; for BSA hydrophobic interactions were suggested to be dominant, whereas in case of LYZ electrostatic interactions contributed the most to the deposition mechanism. The effect of additives of the protein solutions, i.e. inorganic salts, PEG, and urea depended on the adsorption mechanism and was also specific for each protein. Nevertheless, the proposed procedure performed well in the evaluation of surface deposition and yield reduction, regardless of the protein type and its solvent environment. 相似文献
19.
Toxoplasma gondii: ultrastructural localization of specific antigens and inhibition of intracellular multiplication by monoclonal antibodies 总被引:1,自引:0,他引:1
This experiment was focused on the characterization of anti-Toxoplasma monoclonal antibodies (mAbs) and the effect of mAbs on the parasite invasion of mouse peritoneal macrophages. Twenty eight mAbs including M110, M556, R7A6 and M621 were characterized by Ab titer, immunoglobulin isotyping and western blot pattern. Antibody titer (optical density) of 4 mAbs, M110, M556, R7A6 and M621, were 0.53, 0.67, 0.45 and 0.39 (normal mouse serum; 0.19) with the same IgG1 isotypes shown by Enzyme-linked immunosorbent assay (ELISA). Western blot analysis showed that M110, M556, R7A6 and M621 reacted with the 33 kDa (p30), 31 kDa (p28), 43 kDa and 36 kDa protein. Immunogold labelling of mAbs M110, M556, R7A6 and M621 reacted with the surface membrane, dense granules and parasitophorous vacuolar membrane (PVM), rhoptries and cytoplasm of tachyzoite, respectively. For in vitro assay, preincubation of tachyzoites with four mAbs, M110, M556, R7A6 and M621 resulted in the decrease of the number of infected macrophages (p < 0.05) and the suppression of parasite multiplication at 18 h post-infection. Four monoclonal antibodies including M110 (SAG1) were found to have an important role in the inhibition of macrophage invasion and T. gondii multiplication in vitro, and these mAbs may be suitable for vaccine candidates, diagnostic kit and for chemotherapy. 相似文献
20.
Wickramasinghe SR Kalbfuss B Zimmermann A Thom V Reichl U 《Biotechnology and bioengineering》2005,92(2):199-208
Large scale purification of viruses and viral vectors for gene therapy applications and viral vaccines is a major separation challenge. Here tangential flow microfiltration and ultrafiltration using flat sheet membranes has been investigated for concentration of human influenza A virus. Ultrafiltration membranes with molecular weight cutoffs of 100 and 300 kDa as well as 0.1, 0.2 and 0.45 microm microfiltration membranes have been tested. The results indicate that use of 300 kDa membranes not only concentrate the virus particles but also lead to a significant removal of host cell proteins and DNA in the permeate. Tangential flow filtration may be used to fractionate virus particles. Human influenza A virus particles are spherical with an average size of 100 nm. Use of a 0.1 microm membrane leads to passage of virus particles less than 100 nm into the permeate and an increase of larger particles in the retentate. These results suggest that control of the transmembrane pressure, membrane pore size and pore size distribution could enable isolation of intact virus particles from damaged virions. Isolation of the virus particles of interest from viral fragments and other particulate matter could result in simplification of subsequent purification steps. Larger pore size membranes such as 0.45 microm that allow the passage of all virus particles may be used to remove host cell fragments. In addition virus particles attached to these fragments will be removed. Careful selection of membrane morphology and operating conditions will be essential in order to maximize the benefit of tangential flow filtration steps in the purification of viral products from cell cultures. 相似文献