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Biomedical subjects

Manuel J T Carrondo

Publications and source records attributed to Manuel J T Carrondo.

At least 19 recordsLinked to original sources
PubMed2006

Catalase effect on cell death for the improvement of recombinant protein production in baculovirus-insect cell system.

Baculovirus expression vector system (BEVS) in host insect cells is a powerful technology to produce recombinant proteins, as well as virus-like particles (VLP). However, BEVS is based on baculovirus infection, which limits the recombinant protein production by inducing insect cell death. Herein a new strategy to enhance cell life span and to increase recombinant protein production was developed. As baculovirus infection induces cellular oxidative stress, the ability of several antioxidants to inhibit cell death was tested during infection. The production of rotavirus structural proteins was used as model to analyse this new strategy. We found that only catalase is able to partially prevent cell death triggered by baculovirus infection and to inhibit lipid peroxidation. An increase in recombinant protein production was coupled with the partial cell death inhibition. In summary, the addition of catalase is a promising strategy to improve recombinant protein production in BEVS, by delaying insect cell death.

Animals↗
PubMed2006

Purification of retroviral vectors for clinical application: biological implications and technological challenges.

For centuries mankind led a difficult battle against viruses, the smallest infectious agents at the surface of the earth. Nowadays it is possible to use viruses for our benefit, both at a prophylactic level in the production of vaccines and at a therapeutic level in the promising field of gene therapy. Retroviruses were discovered at the end of the 19th century and constitute one of the most effective entities for gene transfer and insertion into the genome of mammalian cells. This attractive feature has intensified research in retroviral vectors development and production over the past years, mainly due to the expectations raised by the concept of gene therapy. The demand for high quality retroviral vectors that meet standard requisites from the regulatory agencies (FDA and EMEA) is therefore increasing, as the technology has moved into clinical trials. The development of safer producer cell lines that can be used in large-scale production will result in the production of large quantities of retroviral stocks. Cost-efficient and scalable purification processes are essential for production of injectable-grade preparations to achieve final implementation of these vectors as therapeutics. Several preparative purification steps already established for proteins can certainly be applied to retroviral vectors, in particular membrane filtration and chromatographic methods. Nevertheless, the special properties of these complex products require technological improvement of the existing purification steps and/or development of particular purification steps to increase productivity and throughput, while maintaining biological activity of the final product. This review focuses on downstream process development in relation to the retroviral vectors characteristics and quality assessment of retroviral stocks for intended use in gene therapy.

Genetic Therapy↗
PubMed2006

Screening anion-exchange chromatographic matrices for isolation of onco-retroviral vectors.

The adsorption kinetics of retroviral vectors to several chromatographic media, DEAE FF, Streamlinetrade mark Q XL and CHTtrade mark Ceramic Hydroxyapatite, in batch mode was investigated. The effects of buffer type, pH and operational temperature were studied. A mathematical model describing viral adsorption kinetics that considers viral degradation in solution was developed. The best results, either in terms of speed and extent of adsorbed infectious particles, were obtained with DEAE FF and Streamlinetrade mark Q XL. Fixed-bed chromatography was further investigated using DEAE FF, Q XL and Q FF, for validation of the batch adsorption process. Fixed-bed DEAE FF and Q XL proved to be good candidates for purification of MoMLV derived vectors due to resulting high yields, 53+/-13% and 51+/-7%, respectively, while removing more than 99% of protein and 90% of the DNA contaminants.

Adsorption↗
PubMed2006

A novel recombinant virus-like particle vaccine for prevention of porcine parvovirus-induced reproductive failure.

A novel vaccine against porcine parvovirus (PPV), composed of recombinant virus-like particles (PPV-VLPs) produced with the baculovirus expression vector system (BEVS) at industrial scale, was tested for its immunogenicity and protective potency. A formulation of submicrogram amounts of PPV-VLPs in a water-in-mineral oil adjuvant evoked high serum antibody titres in both guinea pigs, used as reference model, and target species, pigs. A single immunisation with 0.7microg of this antigen yielded complete foetal protection against PPV infection after challenge with a virulent strain of this virus. Furthermore, also in the presence of mild adjuvants the protective action of these PPV-VLPs is excellent. This recombinant subunit vaccine overcomes some of the drawbacks of classical PPV vaccines.

Adjuvants, Immunologic↗
PubMed2005

Process development for the mass production of Ehrlichia ruminantium.

This work describes the optimization of a cost-effective process for the production of an inactivated bacterial vaccine against heartwater and the first attempt to produce the causative agent of this disease, the rickettsia Ehrlichia ruminantium (ER), using stirred tanks. In vitro, it is possible to produce ER using cultures of ruminant endothelial cells. Herein, mass production of these cells was optimized for stirring conditions. The effect of inoculum size, microcarrier type, concentration of serum at inoculation time and agitation rate upon maximum cell concentration were evaluated. Several strategies for the scale-up of cell inoculum were also tested. Afterwards, using the optimized parameters for cell growth, ER production in stirred tanks was validated for two ER strains (Gardel and Welgevonden). Critical parameters related with the infection strategy such as serum concentration at infection time, multiplicity and time of infection, and medium refeed strategy were analyzed. The results indicate that it is possible to produce ER in stirred tank bioreactors, under serum-free culture conditions, reaching a 6.5-fold increase in ER production yields. The suitability of this process was validated up to a 2-l scale and a preliminary cost estimation has shown that the stirred tanks are the least expensive culture method. Overall, these results are crucial to define a scaleable and fully controlled process for the production of a heartwater vaccine and open "new avenues" for the production of vaccines against other ehrlichial species, with emerging impact in human and animal health.

Animals↗
PubMed2005

Characterization of Ehrlichia ruminantium replication and release kinetics in endothelial cell cultures.

Ehrlichia ruminantium is the causative agent of Heartwater, a fatal tick-borne disease affecting ruminants in African countries and West Indies and can be used as an inactivated vaccine for wild and domestic animals. In order to improve E. ruminantium production yields we characterize E. ruminantium growth kinetics in terms of duplication time, maximum production yield, and peak of infectivity. After a 24 h period for E. ruminantium attachment/internalization and a lag phase of 12 h, the exponential growth occurred within 36-108 h post-infection (hpi) with a net increase of up to 2.2 orders of magnitude. Maximum E. ruminantium infectivity was observed at 120 hpi and was defined as the best time of harvesting (TOH) for propagation of E. ruminantium cultures. This study showed that considering the quality constraint of the final product (E. ruminantium vaccine), the E. ruminantium suspension should be harvested at 113 hpi. Overall, the characterization of E. ruminantium progression through the average infection cycle, not only can contribute to the maximization of E. ruminantium production yield, with important consequences for the large scale production and utilization of an inactivated Heartwater vaccine, but also to elucidate growth mechanisms of some of the other ehrlichial species, with emerging impact in human and animal health.

Animals↗
PubMed2005

Triple layered rotavirus VLP production: kinetics of vector replication, mRNA stability and recombinant protein production.

Rotavirus infection causes diarrhoeal disease in infants, killing more than half million children each year. Virus-like particles (VLP) seem to be excellent vaccine candidates, since they are cheaper to produce than attenuated viral vaccines and safer, as they do not contain genetic material. The present work focus on a triple layered particle composed by three rotavirus structural proteins: VP2, VP6 and VP7, produced in an insect cell/baculovirus expressing system. Two strategies were evaluated for 2/6/7 VLP production: co-infection with three monocistronic baculovirus vectors or single-infection with a tricistronic multi-gene baculovirus vector; these strategies were followed at different levels: baculovirus DNA replication kinetics, mRNA stability, protein production and VLP formation. This study highlights some of the reasons why the tricistronic baculovirus strategy is more efficient for production of triple layered rotavirus 2/6/7 VLP than monocistronic co-infection, in particular: (i) the tricistronic vector presents higher DNA replication rates than the monocistronic vectors, (ii) the mRNA stability is invariant for all mRNAs corresponding to VP2, VP6 and VP7 and (iii) the tricistronic baculovirus strategy produces an excess of VP7 over VP6 when compared to the VP7/VP6 stoichiometric ratio in the native rotavirus. Although the co-infection strategy leads to protein production akin to the rotavirus VP7/VP6 stoichiometric ratio, the tricistronic vector strategy yields higher amounts of rotavirus-like particles.

Animals↗
PubMed2005

Quantification of Ehrlichia ruminantium by real time PCR.

Ehrlichia ruminantium (ER) is the causative agent of Heartwater, one of the most common tick-borne diseases affecting ruminants in African countries and West Indies. Although ER can be used as an inactivated vaccine for wild and domestic animals, there are currently no easy and reliable methods for the quantification of this obligate intracellular bacterium. This report describes the development of a SYBR Green I based real time PCR protocol for the quantification of ER for vaccine production purposes. The method was validated for four ER strains. The external-standard-based PCR protocol developed has a large dynamic quantitative range allowing accurate ER measurement in samples containing from 10(2) to 10(8) gene copies; the method is also reproducible and precise, with intra- and inter-assay coefficients below 5%. The detection limits were validated for samples collected from bovine aortic endothelial cell culture bulks, which are commonly used to produce the ER vaccine. In contrast to the methods based upon protein content, no interference from the host cells in ER quantification was observed. Furthermore, the extended applicability of the new technique was demonstrated by monitoring ER production in cell culture thus rendering it a valuable tool to ensure consistency between vaccine lots and to evaluate optimal vaccine dosage.

Africa South of the Sahara↗
PubMed2005

Cultures of rat astrocytes challenged with a steady supply of glutamate: new model to study flux distribution in the glutamate-glutamine cycle.

Glutamate metabolism in astrocytes was studied using an experimental setup that simulates the role of neurons (glutamate producers and glutamine consumers) by the addition of glutaminase to the culture medium. Thereby, a steady supply of glutamate was imposed at the expense of glutamine, and the stress intensity was manipulated by changing the glutaminase concentration. Glutamate supply rates in the range 8-23 nmol/min/mg protein were examined for periods of up to 48 h. When the glutamate supply rate exceeded the uptake rate of this amino acid, a transient increase in the extracellular concentration of glutamate was observed. In response to this stress, the fluxes through the glutamate transporter and glutamine synthetase were increased considerably, and the extracellular concentration of glutamate was eventually restored to a low level. The increased levels of glutamine synthetase were demonstrated by immunoblotting analysis. The effect on glutamate metabolism of the transaminase inhibitor, aminooxyacetic acid (AOAA), and of NH4Cl was also investigated. The supply of glutamate caused a concomitant reduction in the levels of phosphocreatine, phosphoethanolamine, and phosphocholine without affecting the ATP pool. Glutamine synthetase was shown to be is a key element in the control of glutamate metabolism in astrocytic cultures. The metabolic fate of glutamate depends greatly on the time of endurance to the challenge: in naive cells, glutamate was primarily metabolized through the transaminase pathway, while in well-adapted cells glutamate was converted almost exclusively through glutamine synthetase.

Animals↗
PubMed2005

Two different serum-free media and osmolality effect upon human 293 cell growth and adenovirus production.

Adenoviruses are promising vectors for gene therapy and vaccination protocols. Consequently, the market demands for adenovirus are increasing, driving the search for new methodologies for large-scale production of concentrated vectors with warranted purity and efficacy, in a cost-effective way. Nevertheless, the production of adenovirus is currently limited by the so-called 'cell density effect', i.e. a drop in cell specific productivity concomitant with increased cell concentration at infection. Of two different serum-free culture media (CD293 and EX-Cell), evaluated for their effect on human 293 cells growth and adenovirus production at cell densities higher than 1x10(6) cells/ml, EX-Cell proved the better medium for cell growth. Although adenovirus production was equivalent in both media when the infection was performed at 1x10(6) cells/ml, at 3x10(6) cells/ml CD293 was the better. This result related to the high ammonia content in EX-Cell medium at the highest cell concentration at infection. Besides this, the large-scale production of these vectors at high cell densities often requires re-feed strategies, which increase medium osmolality. While a negative effect on cell growth was observed with increasing osmolalities, adenovirus productivity was only affected for osmolalities higher than 430 mOsm.

Adenoviridae↗
PubMed2004

Scale-up of virus-like particles production: effects of sparging, agitation and bioreactor scale on cell growth, infection kinetics and productivity.

The baculovirus-insect cells expression system was used for the production of self-forming Porcine parvovirus (PPV) like particles (virus-like particles, VLPs) in serum-free medium. At 2l bioreactor scale an efficient production was achieved by infecting the culture at a concentration of 1.5 x 10(6)cells/ml using a low multiplicity of infection of 0.05 pfu per cell. In a continuous bioreactor, it was shown that the uninfected insect cells were not sensitive to local shear stress values up to 2.25 N/m2 at high Reynolds numbers (1.5 x 10(4)) in sparging conditions. Uninfected insect cells can be grown at scaled-up bioreactor at high agitation and sparging rates as long as vortex formation is avoided and bubble entrapment is minimized. An efficient process scale-up to 25 l bioreactor was made using constant shear stress criteria for scale-up. The kinetics of baculovirus infection at low multiplicity of infection, either at different cell concentration or at different scales, are very reproducible, despite the different turbulence conditions present in the bioreactor milieu. The results suggest that the infection kinetics is controlled by the rate of baculovirus-cell receptor attachment and is independent of the bioreactor hydrodynamic conditions. Furthermore, the achieved specific and volumetric productivities were higher at the 25 l scale when compared to the smaller scale bioreactor. Different rates of cell lysis after infection were observed and seem to fully explain both the shift in optimal harvest time and the increase in cell specific productivity. The results emphasize the importance of integrated strategies and engineering concepts in process development at bioreactor stage with the baculovirus insect cell system.

Animals↗
PubMed2003

Virus-like particle production at low multiplicities of infection with the baculovirus insect cell system.

The baculovirus insect cell expression system (BEVS) was used for the production of self-forming Porcine parvovirus-like particles (VLPs) in serum-free medium. A low multiplicity of infection (MOI) strategy was used to overcome an extra virus amplification step, undesirable in industrial production, and to minimize the virus passage effect. It was confirmed that the time of infection (TOI) and MOI are dependent variables. Higher cell densities were obtained at low MOIs, keeping a constant TOI; however, both volumetric and specific productivities were lower. In synchronous infection, at high MOI, the specific productivity decreased when the cells were infected in the late phase of growth. Product degradation due to cell lysis strongly influenced the optimal time of harvest (TOH). Time of harvest was found to be highly dependent on the MOI, and a direct relationship with the cell yield was obtained. Analysis of the culture medium reveals that glutamine depletion occurs in the late phase of the growth. Supplementation of glutamine to uninfected cell cultures resulted in an increased cell yield. Its addition to cultures infected in the middle phase of the growth curve was also able to restore the productivity levels, but addition to cells in their stationary phase caused no observable effect on product expression. The study clearly shows that for a specific TOI it is not obvious what the correct MOI should be to obtain the best volumetric productivity.

Algorithms↗
PubMed2002

Production of core and virus-like particles with baculovirus infected insect cells.

In this paper the fundamental aspects of process development for the production of core and virus-like particles with baculovirus infected insect cells are reviewed. The issues addressed include: particle formation and monomer composition, chemical and physical conditions for optimal cell growth, baculovirus replication and product expression, multiplicity of infection strategy, and scale-up of the process. Study of the differences in the metabolic requirements of infected and non-infected cells is necessary for high cell density processes. In the bioreactor, the specific oxygen uptake rate (OURsp) plays a central role in process scale-up, leading to the specification of the bioreactor operational parameters. Shear stress can also be an important variable for bioreactor operation due to its influence on cell growth and product expression. The determination of the critical variables in process development is discussed, showing the relevance of the mathematical models that have been developed for the insect cells/baculovirus system in process implementation and control.

Animals↗
PubMedDate not supplied

Culturing primary brain astrocytes under a fully controlled environment in a novel bioreactor.

We report the first approach for growth and maintenance of primary astrocytes on a fully controlled environment. For this purpose, cells were immobilized in Cytodex microcarriers and grown in a stirred tank bioreactor. The distribution of astrocytes at the microcarrier surface was visualized using confocal microscopy and glial fibrillary acidic protein (GFAP) labeling, a specific glial probe. Crucial bioreaction parameters such as agitation rate, microcarrier type, and concentration, as well as cell inoculum concentration were assessed. Cytodex 3 proved the best microcarrier for astrocyte growth, with the highest cell densities obtained for 6 g/l of Cytodex 3 using an inoculum of approx. 0.15 x 10(6) cells/ml in vessels operated at 60 rpm, using a refeed operational mode consisting of complete medium replacement every 5 days. Using such optimized conditions, cells were maintained in steady-state for approximately 24 days, allowing online monitoring and control of environmental variables such as temperature, pH, and O(2). To test further the advantages of this fully controlled system, astrocytes were also subjected to hypoxic stress for 5 hr; the cell number was not affected by hypoxia but the glycolytic flux was enhanced during the stress imposed. The culture system described is a novel tool to study brain cell metabolism, allowing sampling over time and the monitoring of cellular behavior through stressful conditions and during recovery.

Animals↗
PubMedDate not supplied

Insect cell culture medium supplementation with fetal bovine serum and bovine serum albumin: effects on baculovirus adsorption and infection kinetics.

The effects of insect cell culture medium supplementation with FBS were investigated. BSA was found to be the factor responsible for the increased baculovirus infection rate of FBS-supplemented cultures in a concentration-dependent form up to 25 g L(-)(1). Lower rates of baculovirus binding to cells were observed with FBS- and BSA-supplemented cultures compared with infections carried out in serum-free media. Virus attachment constants were found to depend on medium matrix composition. An efficiency factor dependent on the medium matrix composition was introduced to account for these effects, and a mathematical model was developed to describe the virus-cell interactions. It was shown that BSA acts by minimizing the nonspecific virus binding leading to an increased cell infection rate. Cell specific Porcine parvovirusvirus-like particles (PPV-VLPs) expression was unaffected by medium supplementation pointing out that BSA and/or FBS affects mainly the initial phase of the baculovirus infection cycle. Implications for process definition are discussed.

Adsorption↗
PubMedDate not supplied

Enhancement of Sf-9 cell growth and longevity through supplementation of culture medium with hemolymph.

The benefits of insect cell culture medium supplementation with hemolymph of Lonomia obliqua were investigated. The addition of hemolymph to the medium induced high levels of cell growth, and the viability was maintained for longer periods. The maximum cell yield increased almost 3-fold after hemolymph supplementation. Cultures in their stationary phase were rescued through hemolymph supplementation, also reaching high cell concentrations. These actions were much dependent on the concentration of hemolymph; low hemolymph concentration had a positive effect in cell growth, whereas high hemolymph concentration showed a deleterious effect. Fractionation of hemolymph by gel filtration chromatography showed the presence of three factors with different activity in insect cell culture: an potential anti-apoptotic factor, a growth-promoting factor, and an enzyme that hydrolyzes sucrose. Addition of hemolymph to the medium induced high levels of glucose production. The sucrose to glucose conversion was also linearly dependent upon the hemolymph concentration. Therefore, we conclude that cell growth and longevity can be increased by supplementation of the culture medium with hemolymph.

Animals↗
PubMedDate not supplied

Cell growth arrest by nucleotides, nucleosides and bases as a tool for improved production of recombinant proteins.

Arresting cell growth and thus decreasing cell division potentially lessens the chance for genetic drift in the cell population; this would be of utmost importance for the consistent production of biopharmaceuticals during long periods. The drawback of the addition of well-known synchronizing agents, such as chemotherapeutics, is that they cause a disproportionate accumulation of cellular constituents, leading to cell death. The use of compounds that are naturally synthesized by the cell, as is the case of nucleotides, nucleosides, and bases (Nt/Ns/B), is shown in this work to be a promising tool. The addition of purines and pyrimidines was tested using a CHO cell line producing the secreted form of the human placental alkaline phosphatase enzyme (SEAP). From the chemical alternatives tested, AMP was the most promising compound for protein production improvement; it reduced cell growth and maintained the culture with high cell viability for long periods, while increasing SEAP specific productivity 3-fold. The use of CHO and BHK mammalian cells producing Factor VII and the use of a insect cell line (Sf9) showed that the effect of AMP addition seems to be independent of the r-protein and cell line. With the addition of AMP, accumulation of cells at the S phase was accompanied by an increase of the protein specific productivity. Addition of known synchronizing drugs (aphidicolin and doxorubicin) and application of environmental cell growth arrest strategies (depletion of nutrients and byproduct accumulation) showed also to effectively arrest CHO cell growth. A careful look onto cell cycle distribution in the different scenarios created, shows whether it is important to consider r-protein expression dependency upon cell cycle in process optimization and operation strategies.

Alkaline Phosphatase↗