Laura A. Palomares
National Autonomous University of Mexico
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Featured researches published by Laura A. Palomares.
Methods of Molecular Biology | 2004
Laura A. Palomares; Sandino Estrada-Moncada; Octavio T. Ramírez
Efficient strategies for the production of recombinant proteins are gaining increasing importance, as more applications that require high amounts of high-quality proteins reach the market. Higher production efficiencies and, consequently, lower costs of the final product are needed for obtaining a commercially viable process. In this chapter, common problems in recombinant protein production are reviewed and strategies for their solution are discussed. Such strategies include molecular biology techniques, as well as manipulation of the culture environment. Finally, specific problems relevant to different hosts are discussed.
Molecular Biotechnology | 2006
Alvaro R. Lara; Enrique Galindo; Octavio T. Ramírez; Laura A. Palomares
The presence of spatial gradients in fundamental culture parameters, such as dissolved gases, pH, concentration of substrates, and shear rate, among others, is an important problem that frequently occurs in large-scale bioreactors. This problem is caused by a deficient mixing that results from limitations inherent to traditional scale-up methods and practical constraints during large-scale bioreactor design and operation. When cultured in a heterogeneous environment, cells are continuously exposed to fluctuating conditions as they travel through the various zones of a bioreactor. Such fluctuations can affect cell metabolism, yields, and quality of the products of interest. In this review, the theoretical analyses that predict the existence of environmental gradients in bioreactors and their experimental confirmation are reviewed. The origins of gradients in common culture parameters and their effects on various organisms of biotechnological importance are discussed. In particular, studies based on the scale-down methodology, a convenient tool for assessing the effect of environmental heterogene ities, are surveyed.
Cytotechnology | 1996
Laura A. Palomares; Octavio T. Ramírez
AbstractDissolved oxygen tension and oxygen uptake rate are critical parameters in animal cell culture. However, only scarce information of such variables is available for insect cell culture. In this work, the effect of dissolved oxygen tension (DOT) and the utility of on-line oxygen uptake rate (OUR) measurements in monitoring Spodoptera frugiperda (Sf9) cultures were determined. Sf9 cells were grown at constant dissolved oxygen tensions in the range of 0 to 30%. Sf9 metabolism was affected only at DOT below 10%, as no significant differences on specific growth rate, cell concentration, amino acid consumption/production nor carbohydrates consumption rates were found at DOT between 10 and 30%. The specific growth rate and specific oxygen uptake rate followed typical Monod kinetics with respect to DOT. The calculated μmax and
Enzyme and Microbial Technology | 2000
Laura A. Palomares; Miranda González; Octavio T. Ramírez
Biotechnology Progress | 2003
Laura A. Palomares; Christoph E. Joosten; Patrick R. Hughes; Robert R. Granados; Michael L. Shuler
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Journal of Biotechnology | 1999
Ronaldo Z. Mendonça; Laura A. Palomares; Octavio T. Ramírez
Biotechnology Letters | 2001
Laura A. Palomares; Juan Carlos Pedroza; Octavio T. Ramírez
max were 0.033 h-1 and 3.82×10-10 mole cell-1h-1, respectively, and the corresponding saturation constants were 1.91 and 1.57%, respectively. In all aerated cultures, lactate was consumed only after glucose and fructose had been exhausted. The yield of lactate increased with decreasing DOT. It is proposed, that an ‘apparent’ DOT in non-instrumented cultures can be inferred from the lactate yield of bioreactors as a function of DOT. Such a concept, can be a useful and important tool for determining the average dissolved oxygen tension in non-instrumented cultures. It was shown that the dynamic behavior of OUR can be correlated with monosaccharide (fructose and glucose) depletion and viable cell concentration. Accordingly, OUR can have two important applications in insect cell culture: for on-line estimation of viable cells, and as a possible feed-back control variable in automatic strategies of nutrient addition.
Microbial Cell Factories | 2011
William A. Rodríguez-Limas; Keith E.J. Tyo; Jens Nielsen; Octavio T. Ramírez; Laura A. Palomares
Pluronic F-68 has been widely used to protect animal cells from hydrodynamic stress, but its mechanism of action is still debatable. Published evidence indicates that Pluronic F-68 interacts with cells, yet scarce information exists of its effect on recombinant protein and virus production by insect cells. In this work, the effect of Pluronic F-68 on production of recombinant baculovirus and rotavirus protein VP7 was determined. Evidence of Pluronic F-68 direct interaction with Sf-9 insect cells also was obtained. Maximum recombinant VP7 concentration and yield increased 10x, whereas virus production decreased by 20x, in spinner flask cultures with 0.05% (w/v) Pluronic F-68 compared to controls lacking the additive. No differences were observed in media rheology, nor kinetics of growth and infection (as inferred from cell size) between both cultures. Hence, Pluronic F-68 influenced cell physiology independently of its shear protective effect. Cells subjected to a laminar shear rate of 3000 s(-1) for 15 min, without gas/liquid interfaces, were protected by Pluronic F-68 even after its removal from culture medium. Furthermore, the protective action was immediate in vortexed cells. The results shown here indicate that Pluronic F-68 physically interacts with cells in a direct, strong, and stable mode, not only protecting them from hydrodynamic damage, but also modifying their capacity for recombinant protein and virus production.
Biotechnology Progress | 2008
Matthew L. Lipscomb; Laura A. Palomares; Vanessa Hernández; Octavio T. Ramírez; Dhinakar S. Kompala
Paucimannose or oligomannose structures are usually attached to glycoproteins produced by insect cells, while mammalian glycoproteins usually have complex glycans. The lack of complex glycosylation has limited the use of the insect cell baculovirus expression vector system (BEVS), despite its high productivity and versatility. The availability of cell lines capable of complex glycosylation can overcome such a problem and potentially increase the utility of BEVS. In this work the capability of two novel cell lines, one from Pseudaletia unipuncta (A7S) and one from Danaus plexippus (DpN1), to produce and glycosylate a recombinant protein (secreted human placental alkaline phosphatase, SeAP) was assessed. SeAP produced by Tn5B1–4 cells at a low passage number (<200) was utilized for comparison. The optimal conditions for the production of SeAP by DpN1 cells were defined, and the glycosylation profiles of SeAP produced by the cell lines were quantitatively determined. Both the A7S and the DpN1 cells produced lower concentrations of SeAP than the Tn5B1–4 cells. Less than 5% of the glycans attached to SeAP produced by the Tn5B1–4 cells had complex forms. Glycans attached to SeAP from A7S cells contained 4% hybrid and 8% complex forms. Galactosylated biantennary structures were identified. Glycans attached to SeAP produced by the DpN1 cell line had 6% hybrid and 26% complex forms. Of the complex forms in SeAP from DpN1, 13% were identified as sialylated glycans. The galactosyltransferase activity of the three cell lines was measured and correlated to their ability to produce complex forms. Even though neither novel cell line produced as much recombinant protein as the Tn5B1–4 cells, the glycosylation of SeAP expressed by both cell lines was more complete. These novel cell lines represent interesting alternatives for the production of complex glycosylated proteins utilizing the BEVS.
Biotechnology and Applied Biochemistry | 2007
J. Antonio Serrato; Vanessa Hernández; Sandino Estrada-Mondaca; Laura A. Palomares; Octavio T. Ramírez
Nutritional information of insect cell cultures, required for designing strategies to increase protein productivity, is still limited. In this work, nutrient feeding or removal was used to determine the role of selected nutrients and their interactions in insect cell metabolism before viral infection. Glucose, glutamine, tyrosine, and methionine were consumed at the highest rates in batch and fed-batch cultures, and thus were selected to be manipulated. Glucose and glutamine could be replaced by other carbon sources or amino acids, respectively. However, growth was reduced when such alternative sources were utilized, revealing the distinctive role of glucose and glutamine. Glucose and glutamine consumption rates were regulated by their own concentration, although glutamine concentration did not affect glucose consumption rate or vice versa. Excessive glucose or glutamine supply caused energy and carbon wastes, as observed by reduced cell yields and accumulation of by-products, such as alanine. Nevertheless, growth inhibition was attenuated by simultaneous excess of both nutrients, probably because cells had both carbon and ammonia available to deal with toxic by-products in the form of alanine. Other amino acids, such as tyrosine and methionine, were also relevant for maintaining prolonged viability. The importance of an adequate nutrient supply was demonstrated; particularly, the need to maintain glucose and glutamine concentrations just above their critical values.