RESUMEN
Virus retention during ultrafiltration through A/G Technology filter cartridges was investigated to characterize the removal process and validate the degree of virus titre reduction during the filtration of red blood cell haemolysates performed as part of the production of diaspirin crosslinked haemoglobin (DCLHb). When viruses were suspended in phosphate buffered saline solution, retention was greater with larger sized viruses and smaller filter pore size. Virus titre was maintained at starting levels in the filter retentate circuit during the course of filtration, suggesting that the virus removal mechanism is predominantly size exclusion. Evaluation of specific processing variables indicated that the retention of phiX174 virus was increased in the presence of red blood cell haemolysate or at high membrane crossflow rates and transmembrane pressures, while the retention of EMC virus was less sensitive to variations in these parameters. Using these results to design a validation protocol, log reduction values of >7.9 were demonstrated for the retention of human immunodeficiency virus, pseudorabies virus and bovine viral diarrhoea viruses, 7.6 for hepatitis A virus, and 4.2 for porcine parvovirus. It was also shown that the retention of viruses was maintained during repetitive use of the same filter cartridge.
Asunto(s)
Aspirina/análogos & derivados , Contaminación de Medicamentos , Hemoglobinas/aislamiento & purificación , Ultrafiltración , Virus , Animales , Aspirina/aislamiento & purificación , Bacteriófago phi X 174 , Línea Celular , Virus de la Diarrea Viral Bovina , Virus de la Encefalomiocarditis , Diseño de Equipo , Eritrocitos , Estudios de Evaluación como Asunto , VIH , Hemólisis , Hepatovirus , Herpesvirus Suido 1 , Humanos , Macaca mulatta , Membranas Artificiales , Tamaño de la Partícula , Parvovirus , Seguridad , Porcinos , Ultrafiltración/instrumentación , Ensayo de Placa ViralRESUMEN
Assay validation is the characterization of assay performance that allows the significance of the values obtained in an assay to be evaluated. Biological assays have several inherent sources of variation and this has been used as an excuse for years to explain unexpected or unanticipated results. However, with careful control of reagents and strictly controlled procedures, biological assays can be successfully validated to allow accurate quantitation and interpretation of the result obtained.
Asunto(s)
Bioensayo/métodos , Virología/métodos , Virus/aislamiento & purificación , Animales , Bioensayo/normas , Bioensayo/estadística & datos numéricos , Humanos , Control de Calidad , Reproducibilidad de los Resultados , Estados Unidos , United States Food and Drug Administration , Virología/normas , Virología/estadística & datos numéricosRESUMEN
Two experiments were performed to assess viral inactivation during the crosslinking and heat treatment steps of the DCLHb manufacturing process. Stroma free hemoglobin (SFHb) collected from a large scale manufacturing lot was tested in a 1:680 scaled down system in which the key parameters used in the manufacturing process were replicated. In the first study Porcine Parvovirus (PPV), a non-enveloped virus, was used to assess inactivation, while in the second study Bovine Viral Diarrhea Virus (BVDV), an enveloped virus, was utilized. In both experiments, the SFHb solution was deoxygenated and an aliquot of virus suspension was added. To initiate the crosslinking reaction, a solution of bis (3,5-dibromosalicyl) fumarate (DBBF) in HEPES buffer was added to the test solution. In both experiments the reaction times and the degree of crosslinking were normal. After crosslinking, the reaction mixtures were heated to 74 +/- 1 degrees C over 30 minutes, held at 74 +/- 1 degrees C for 90 minutes, and cooled to less than 10 degrees C over 30 minutes. In each experiment the degree of crosslinking of final product was 100% and yield of hemoglobin recovery was normal. Samples were removed prior to crosslinking, after crosslinking and before, during and after heat treatment for determination of virus titer and evaluation of key process parameters. The results from these experiments were consistent with those obtained from the full scale manufacturing process for the deoxygenation, crosslinking and the heat treatment step during the production of DCLHb. The results of virus assays showed that crosslinking has no effect on viruses and their subsequent inactivation by heat treatment.
Asunto(s)
Aspirina/análogos & derivados , Reactivos de Enlaces Cruzados/metabolismo , Virus de la Diarrea Viral Bovina/crecimiento & desarrollo , Hemoglobinas/metabolismo , Calor , Parvovirus/crecimiento & desarrollo , Ensayo de Placa Viral , Animales , Aspirina/metabolismo , Bovinos , PorcinosRESUMEN
Studies of human TSH (hTSH) structure and function have been limited by difficulties in producing large quantities of recombinant hormone. We describe a system for the stable expression of high levels of recombinant human TSH (rec hTSH) using a mutant form of dihydrofolate reductase (dhfr) as an amplifiable dominant selectable marker. A vector expressing both the hTSH alpha-subunit and the mutant dhfr was cotransfected with a hTSH beta-subunit expression vector into dhfr-deficient cells. Amplification of the transfected sequences by methotrexate selection, followed by cell culture in a hollow fiber perfusion system, yielded rec hTSH production as high as 100,000 microU/ mL. Immunoradiometric assays using five different antibodies revealed no differences in the immunological activities of rec hTSH and pituitary hTSH. Bioactivity was measured in a novel TSH bioassay coupling the generation of cAMP by a transfected hTSH receptor to the cAMP-dependent regulation of a luciferase reporter gene. The ED50 for bovine TSH in this bioassay was 1.4 ng/mL (3.5 x 10(-11) mol/L). The ratio of the ED50 values for rec hTSH and pituitary hTSH was 1.0:1.1 (P = NS), indicating that the two TSHs were of equivalent potency. In conclusion, we have developed techniques for the high level production of rec hTSH that is immunologically and biologically equivalent to pituitary hTSH. The ability to produce large quantities of rec hTSH using standard laboratory techniques should facilitate future studies, such as the development of clinically useful TSH analogs.
Asunto(s)
Metotrexato/farmacología , Tirotropina/biosíntesis , Animales , Bioensayo , Células CHO/metabolismo , Bovinos , Cromatografía , Cricetinae , Humanos , Técnicas Inmunológicas , Proteínas Recombinantes , TransfecciónRESUMEN
The ability of purified plasma membrane glycoconjugates to inhibit the EDTA-resistant agglutination between aggregation-stage cells of Dictyostelium discoideum has suggested that receptor binding of these glycoconjugates provides a basis for cell-cell cohesion during aggregation. This has been tested by analysis of a series of mutants with different defects in the assembly of N-linked oligosaccharides. Mutant HL241 lacks outer branch components of N-linked oligosaccharides and fails to aggregate or express EDTA-resistant cohesion. HL244 makes unsulphated but otherwise normal N-linked oligosaccharides, generates multiple tips on aggregated cell mounds in some clones, and shows abnormally strong EDTA-resistant cohesion. Two mutants that are temperature-sensitive for complete processing of N-linked oligosaccharides are also temperature-sensitive for expression of both aggregation ability and EDTA-resistant cohesion. A revertant that recovered essentially normal N-linked oligosaccharide processing at the restrictive temperature has also recovered its ability to aggregate and to agglutinate in EDTA.
Asunto(s)
Dictyostelium/fisiología , Glucolípidos/fisiología , Glicoproteínas de Membrana/fisiología , Oligosacáridos/metabolismo , Aglutinación , Animales , Secuencia de Carbohidratos , Membrana Celular/fisiología , Glucolípidos/química , Glucolípidos/aislamiento & purificación , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/aislamiento & purificación , Lípidos de la Membrana/química , Lípidos de la Membrana/aislamiento & purificación , Lípidos de la Membrana/fisiología , Datos de Secuencia Molecular , Oligosacáridos/química , Oligosacáridos/aislamiento & purificaciónRESUMEN
Mutants of Dictyostelium discoideum have been isolated by a selection for cells with temperature-sensitive defects in the maturation of glycoprotein N-linked oligosaccharides. Here we describe a mutant, HT7, which is unable to aggregate at the restrictive temperature, but which aggregates and makes fruiting bodies at the permissive temperature. HT7 shows normal early developmental intercellular cohesion, but is temperature sensitive for expression of the ethylenediamine-tetraacetic acid (EDTA)-resistant cohesion characteristic of aggregation. The mutant initiates aggregation, but forms only loose cell mounds which later disperse. Metabolic labelling studies indicate that the thermolabile defect is not in protein synthesis, assembly of the lipid-linked precursor of N-linked oligosaccharides or transfer of the precursor to proteins. However, the defect does prevent assembly of fully processed N-linked oligosaccharides. Further, two glycopeptides, obtained from exhaustive Pronase digests of wild-type plasma membrane glycoproteins, inhibit intercellular cohesion of aggregation-stage wild-type cells. HT7 produces only approximately 50% of the wild-type level of these glycopeptides at the restrictive temperature and one of the glycopeptides has reduced cohesion inhibition ability. A revertant of HT7 was found to aggregate normally, to have restored EDTA-resistant cohesion, to have normal profiles of N-linked oligosaccharides and to express the two cohesion-inhibiting glycopeptides normally. These data strongly support a model in which cohesion during late aggregation is at least in part due to recognition between surface glycans and receptors on neighbouring cells.
Asunto(s)
Dictyostelium/genética , Glicoproteínas/biosíntesis , Aglutinación , Secuencia de Aminoácidos , Animales , Secuencia de Carbohidratos , AMP Cíclico/metabolismo , Dictyostelium/fisiología , Fucosa/metabolismo , Glucolípidos/biosíntesis , Glucolípidos/aislamiento & purificación , Glicoproteínas/genética , Glicoproteínas/aislamiento & purificación , Glicosilación , Manosa/metabolismo , Datos de Secuencia Molecular , Mutación , Sulfatos/metabolismo , Radioisótopos de Azufre , Temperatura , TritioRESUMEN
Human lysosomal beta-hexosaminidase exists in two major forms: the A isoform is composed of both alpha and beta chains, while the B form is a homopolymer of beta chains. Deficiency of beta-hexosaminidase underlies the GM2 gangliosidoses. We have produced active beta-hexosaminidase B in cultured insect (Sf9) cells by isolation of a recombinant insect virus (baculovirus) containing the cDNA for the beta chain within the viral polyhedron gene and infection of Sf9 cells with this construct. That portion of the enzyme secreted into the medium, 50%, was purified with concanavalin A Sepharose and subsequent affinity chromatography to yield beta-hexosaminidase B that is 75% pure. The product has an N-terminal amino acid sequence, specific activity, and size (M(r) 62,000) similar to that of the enzyme present in cultured human fibroblasts. However, endo H sensitivity studies revealed that the oligosaccharide structures present on recombinant beta-hexosaminidase B differ from those found on the enzyme synthesized in the human system. In addition, these structures lack the mannose 6-phosphate recognition marker that targets degradative hydrolases to lysosomes. Despite these differences, recombinant beta-hexosaminidase B does serve as a specific substrate for the mannose phosphorylating enzyme, N-acetylglucosaminyl phosphotransferase. Furthermore, the oligosaccharide moieties phosphorylated in vitro match those phosphorylated in vivo, pointing to the conformational integrity of the recombinant enzyme. Generous amounts of easily obtained, easily purified, and properly folded beta-hexosaminidase B will facilitate physical structural analysis of the enzyme.
Asunto(s)
Transferasas (Grupos de Otros Fosfatos Sustitutos) , beta-N-Acetilhexosaminidasas/biosíntesis , Baculoviridae/genética , Clonación Molecular , ADN/genética , Humanos , Lisosomas/enzimología , Manosafosfatos/química , Peso Molecular , Fosfotransferasas/metabolismo , Conformación Proteica , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Especificidad por Sustrato , Tripsina , beta-N-Acetilhexosaminidasas/genética , beta-N-Acetilhexosaminidasas/aislamiento & purificaciónRESUMEN
Tissue-type plasminogen activator (t-PA), the serine protease responsible for catalyzing the production of plasmin from plasminogen at the site of blood clots, is synthesized as a single-chain polypeptide precursor. Proteolytic cleavage at the C-terminal side of Arg275 generates a two-chain form of the enzyme whose subunits are held together by a single disulfide bond. We have measured the activities of both forms of the wild-type enzyme, as well as that of a mutant enzyme (Arg275----Gly), created by oligonucleotide-directed mutagenesis, that cannot be cleaved into a two-chain form. Both types of single-chain t-PAs are enzymatically active and exhibit identical Vmax and Km values when assayed with synthetic peptide substrates, indicating that the single amino acid change had no effect on the amidolytic activity of the enzyme. However, cleavage of wild-type t-PA into the two-chain form results in increased activity both on a peptide substrate and on the natural substrates Lys- and Glu-plasminogen in the absence or presence of stimulation by soluble fibrin. The enhanced activity is due to a 3-5-fold increase in the Vmax of the cleaved enzyme, rather than to any change in the Km values for the various substrates. During incubation with plasminogen, the single-chain form of wild-type t-PA is converted to the two-chain form by plasmin generated during the reaction. This conversion, from the less active form of the enzyme, results in a reaction that displays biphasic kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Mutación , Activador de Tejido Plasminógeno/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , ADN Recombinante/metabolismo , Humanos , Indicadores y Reactivos , Cinética , Sustancias Macromoleculares , Datos de Secuencia Molecular , Activador de Tejido Plasminógeno/genéticaRESUMEN
The heavy chain of tissue plasminogen activator (t-PA) consists of four domains [finger, epidermal-growth-factor (EGF)-like, kringle 1 and kringle 2] that are homologous to similar domains present in other proteins. To assess the contribution of each of the domains to the biological properties of the enzyme, site-directed mutagenesis was used to generate a set of mutants lacking sequences corresponding to the axons encoding the individual structural domains. The mutant proteins were assayed for their ability to hydrolyze artificial and natural substrates in the presence and absence of fibrin, to bind to lysine-Sepharose and to be inhibited by plasminogen activator inhibitor-1. All the deletion mutants exhibit levels of basal enzymatic activity very similar to that of wild-type t-PA assayed in the absence of fibrin. A mutant protein lacking the finger domain has a 2-fold higher affinity for plasminogen than wild-type t-PA, while the mutant that lacks both finger and EGF-like domains is less active at low concentrations of plasminogen. Mutants lacking both kringles neither bind to lysine-Sepharose nor are stimulated by fibrin. However, mutants containing only one kringle (either kringle 1 or kringle 2) behave indistinguishably from one another and from the wild-type protein. We conclude that kringle 1 and kringle 2 are equivalent in their ability to mediate stimulation of catalytic activity by fibrin.
Asunto(s)
Fragmentos de Péptidos/genética , Activador de Tejido Plasminógeno/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN/genética , Factor de Crecimiento Epidérmico/genética , Exones , Humanos , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Señales de Clasificación de Proteína/genéticaRESUMEN
In order to identify the biological roles of protein-linked oligosaccharides, we have isolated mutants by a selection for amoebae with temperature-sensitive defects in glycan assembly and processing. Of these, 75% were also temperature sensitive for development [Boose and Henderson, 1986]. Two such mutants with distinct developmental phenotypes and glycosylation patterns are described. Mutant HT7 cannot complete aggregation at the restrictive temperature and is defective in expression of EDTA-resistant cohesion. The biochemical defect appears to be early in glycan processing. A revertant of HT7 has recovered aggregation capability, EDTA-resistant cohesion, and reverted almost totally to wild-type glycosylation. Mutant HT15 aggregates at the restrictive temperature but then disperses into a cell lawn. It is less deficient in EDTA-resistant cohesion than HT7 and has a different glycosylation profile. These results provide strong support for a role of protein N-linked oligosaccharides in aggregation-stage intercellular cohesion.
Asunto(s)
Adhesión Celular , Dictyostelium/genética , Mutación , Aglutinación , Membrana Celular/metabolismo , Dictyostelium/fisiología , Glicopéptidos/aislamiento & purificación , Glicosilación , Cinética , Glicoproteínas de Membrana/aislamiento & purificación , Oligosacáridos/análisisRESUMEN
The assembly and processing of glycoprotein-linked oligosaccharides in Dictyostelium discoideum has been shown to generate a wide array of glycan structures which undergo dramatic developmental regulation. As late steps in processing of these oligosaccharides involve sulfation, a sulfate suicide selection procedure was developed to select for temperature-sensitive glycoprotein-processing mutants. Of 673 clones derived from the survivors of suicide selection, 99 were classified by replica-plating fluorography as temperature sensitive for sulfate transport or incorporation. Of these, 74 were unable to complete the developmental program to the fruiting body stage at the restrictive temperature, 29 being blocked in some aspect of aggregation and 45 being blocked at some postaggregation stage. Quantitative metabolic labeling experiments with representative clones showed that they incorporated wild-type levels of [35S]methionine but reduced levels of sulfate at the restrictive temperature. The specific incorporation patterns in the mutants suggest that distinct oligosaccharide-processing steps are involved in different developmental events.