Biomedical subjects

J J Cairó

Publications and source records attributed to J J Cairó.

12 recordsLinked to original sources

PubMed2006

Application of on-line OUR measurements to detect actions points to improve baculovirus-insect cell cultures in bioreactors.

The continuous monitoring of a process based on the culture of Sf9 insect cells and infection by a baculovirus as a vector to obtain recombinant VP2 protein is studied. On-line OUR determination is based on the direct oxygen measurement in the cell culture vessel and the application of the dynamic method. This approximation allows a proper description of cell growth, with precise identification of the balanced cell growth end and the most important action times in the process, as virus infection time and final cell harvesting. A detailed study of the OUR profiles allows on-line monitoring of the effects of infection and expression protein process, a tool enabling the automatisation of the protein production process in a baculovirus-insect cell system. These parameters have been defined as time of action (TOAs), and include the most relevant actions to take in these type of processes: time of infection (TOI), time of feeding (TOF) and time of harvesting (TOH).

Animals↗

PubMed2005

On-line monitoring of yeast cell growth by impedance spectroscopy.

The application of impedance spectroscopy to estimate on-line cell concentration was studied. The estimation was based on the relative variation between electrical impedance measured at low (10 kHz) and high frequencies (10 MHz). Studies were carried out to characterise the influence of changes in physical and chemical parameters on the impedance measurement. Two different possibilities to perform on-line measurements were tested: a simple set-up, based on an in situ probe, gave good results but was not suitable for high agitation and aeration rates. An ex situ flow-through on-line measuring cell was used to overcome these problems, showing a better performance. The use of this set-up for the growth monitorisation of a Saccharomyces cerevisiae culture showed an efficient performance, having the correlation between estimated and measured S. cerevisiae a Pearson coefficient of 0.999.

Biomass↗

PubMed2004

Strategies for fed-batch cultivation of t-PA producing CHO cells: substitution of glucose and glutamine and rational design of culture medium.

A strategy for fed-batch cultivation of t-PA producing recombinant CHO cells is presented, based on the substitution of glucose and glutamine for slowly metabolized nutrients and in a rational design of the medium. Media for the batch and fed stages were based on the cell specific amino acid requirements, which allowed a more accurate determination of the initiation of the fed stage and the frequency of nutrient addition from then on. Salt concentration was also reduced in both media to avoid an increase in osmolality. As a consequence of this rational design, most amino acid did not accumulate significantly during the fed stage, as usually occurs when their supply is not based on cell requirements; also, lower amounts of by-products were obtained when osmolality level was kept low, that altogether increased viability, longevity and t-PA production when compared with a reference batch culture. Alternating glucose and galactose during the fed stage, allowed lactate detoxification of the cells through their own metabolism. This allowed an increase in cell growth and cell viability with respect to a fed-batch culture in which only glucose was used in the fed stage.

Animals↗

PubMed2001

Decoupling cell growth and product formation in Chinese hamster ovary cells through metabolic control.

The development of a strategy for the culture of Chinese hamster ovary (CHO) cells producing tissue plasminogen activator (t-PA) is investigated. This strategy is based on the replacement of the main carbon source, glucose, by another compound that is slowly metabolizable, particularly galactose. The introduction of this change allows for acute change in cell behavior at various levels. Cell growth is stopped after this nutrient shift, and the cells can be kept in long-duration culture at a low growth rate and high viability as compared with a culture strategy based solely on glucose utilization. Moreover, the capability of cells to produce recombinant proteins (t-PA in this work) can be maintained over the entire period of galactose feeding. From the metabolic point of view, use of a slowly metabolizable carbon source (galactose) introduces important changes in the production of lactate, ammonia, and some amino acids. The use of this metabolic shift enables the generation of biphasic processes, with a first phase with cell growth on glucose and a second stationary phase on galactose, which is particularly suited to perfusion systems.

Amino Acids↗

PubMed1994

Determination of ammonium and L-glutamine in hybridoma cell cultures by sequential flow injection analysis.

A flow injection analytical system based on a gas diffusion membrane module for ammonia and an ammonium flow-through potentiometric detector has been set up for measurement of L-glutamine and ammonium ions in hybridoma cell cultures. The main feature of the system is that the same basic analytical concept and equipment is used in both measurements, the only difference being for the determination of L-glutamine, in which the sample flows through an immobilized glutaminase cartridge. The conditions to enable the performance of both analysis consecutively, avoiding potential interferences by unwanted deamination of other compounds in the samples, have been determined. Finally, the proposed system has been compared with reference analytical methods for batch hybridoma cell culture experiments.

Animals↗

PubMed1993

Cyclic disulfide model of the major antigenic site of serotype-C foot-and-mouth disease virus. Synthetic, conformational and immunochemical studies.

A cyclic disulfide peptide representing antigenic site A of foot-and-mouth disease virus (FMDV) strain C-S8c1 (residues 134 to 155 of viral protein 1 (VP1) with Tyr136 and Arg153 replaced by cystine; TTCTASARGDLAHLTTTHACHL) was synthesized by solid phase methods. Formation of the cyclic disulfide was carried out by air oxidation of the fully deprotected and reduced bis-cysteine precursor, under high dilution conditions. The identity of the cyclic peptide was confirmed by both physical and enzymatic methods. A conformational study of the cyclic peptide and of its linear parent structure (YTASARGDLAHLTTTHARHLP, residues 136-156 of VP1 of FMDV C-S8c1) by circular dichroism in the presence of a structure-inducing solvent showed the cyclic disulfide analog to adopt lower levels of alpha-helix than its linear counterpart. In competitive ELISA assays both peptides reacted with similar affinity against a representative panel of neutralizing monoclonal antibodies directed towards antigenic site A. Thus, a high inherent flexibility of this loop may preclude a conformational restriction strong enough to alter recognition by anti-virus antibodies.

Amino Acid Sequence↗

PubMed1993

Substitution of transferrin by FeCl3 in the development of a low foetal calf serum concentration medium for KB-26.5 hybridoma cell line.

KB-26.5, a murine hybridoma cell line producing an IgG3 monoclonal antibody used in blood type determination, primarily adapted to grow at 5% foetal calf serum (FCS) concentration has been adapted to grow at 0.5% FCS, maintaining its ability to produce antibodies at the same level. In the final step of adaptation, the addition of insulin, transferrin, ethanolamine and selenium to the media formulation was studied, using factorial assay techniques to check the effect of the different compounds and to optimize their required level for satisfactory growth and antibody secretion. KB-26.5 cells required only 20 micrograms/ml of transferrin to adapt to 0.5% FCS medium. Furthermore, transferrin could be substituted by FeCl3, at a relatively low level of 2 micrograms/ml. Maximum cell density decreased by 31.5% in spinner flask test, but the antibody titer was maintained, thus the specific productivity increased. However, inoculum size had to be increased three-fold with 0.5% FCS medium in order to assure cell growth.

Adaptation, Physiological↗

PubMed1992

Non-additive effects of multiple amino acid substitutions on antigen-antibody recognition.

Synthetic peptides have been used to mimic the main antigenic site of foot-and-mouth disease virus (FMDV) of serotype C and of several variant isolates. This region includes multiple continuous B cell epitopes. The effect of single amino acid replacements, individually or in combination, on antigen specificity has been evaluated using monoclonal antibodies. Quantitative enzyme immunodot assays have shown that both additive and non-additive effects of multiple replacements occur in continuous B cell epitopes, with regard to antibody recognition. Antigenically critical single replacements may be compensated by other, non-critical replacements. Thus, the role of a single amino acid on antibody recognition depends on the sequence context in the antigenic domain. The non-additive effects of multiple replacements may modulate the extent of antigenic diversification of highly variable RNA viruses, and keep viruses confined within antigenic groups by precluding linear antigenic divergence.

Amino Acid Sequence↗

PubMed1992

Methanosarcina mazei JC2, a new methanogenic strain isolated from lake sediments, that does not use H2/CO2.

A new mesophilic methanogenic strain, which produced methane from acetate, methanol, and methylamines, was isolated from lake sediments obtained from the lake Banyoles, near Girona (Spain). The cells were irregular in shape, from 1 to 3 micron in diameter, aggregated in masses of a few to several hundred units. Colonies were about 1-2 mm and irregularly shaped. Their color was yellow or white. Growth occurred throughout the pH range of 5 to 9 with optimal growth around pH 7. The optimal growth temperature was 37 degrees C. The molar deoxiribonucleic acid base composition was 37.2% (G + C). Studies of DNA homologies showed that this isolated was a strain of Methanosarcina mazei, but it differs from other reported strains, in that was not able to use H2/CO2 for growth or methane production.

Acetates↗

PubMedDate not supplied

Solution versus solid-phase cyclization strategies for large sidechain lactam-bridged peptides: a comparative study.

A 22-residue peptide with a sidechain lactam bridge involving 18 residues (60-atom cycle) has been synthesized. Three different protection schemes using Fmoc/tBu/cyclohexyl, Fmoc/tBu/allyl or Boc/Bzl/ fluorenylmethyl protecting group combinations have been explored for the solid phase of the linear precursors, which have been subsequently cyclized in solution or in the solid phase. Cyclization yields in solution have been consistently better than on solid phase; however, the solid-phase strategy requires fewer purification steps and therefore global yields are comparable.

Amino Acid Sequence↗

PubMedDate not supplied

Analysis of CHO cells metabolic redistribution in a glutamate-based defined medium in continuous culture.

The effect of glutamine replacement by glutamate and the balance between glutamate and glucose metabolism on the redistribution of t-PA-producing recombinant CHO cells metabolism is studied in a series of glucose shift down and shift up experiments in continuous culture. These experiments reveal the existence of multiple steady states, and experimental data are used to perform metabolic flux analysis to gain a better insight into cellular metabolism and its redistribution. Regulation of glucose feed rate promotes a higher efficiency of glucose and nitrogen source utilization, with lower production of metabolic byproducts, but this reduces t-PA specific production rate. This reduction under glucose limitation can be attributed to the fact that the cells are forced to efficiently utilize the carbon and energy source for growth, impairing the production of dispensable metabolites. It is, therefore, the combination of growth rate and carbon and energy source availability that determines the level of t-PA production in continuous culture.

Adenosine Triphosphate↗

PubMedDate not supplied

Improvement of CHO cell culture medium formulation: simultaneous substitution of glucose and glutamine.

The formulation of the culture medium for a Chinese hamster ovary (CHO) cell line has been investigated in terms of the simultaneous replacement of glucose and glutamine, the most commonly employed carbon and nitrogen sources, pursuing the objective of achieving a more efficient use of these compounds, simultaneously avoiding the accumulation of lactate and ammonium in the medium. The key factor in this process is the selection of compounds that are slowly metabolized. Among the different compounds studied, galactose and glutamate provide the best results, allowing support of cell growth with an optimal balance between nutrient uptake and cell requirements and the generation of minimal quantities of lactate and ammonium. The attained results also highlight the capacity of the cells to redistribute their metabolism as a response to the changes in medium composition.

Animals↗