Biomedical subjects

J A Asenjo

Publications and source records attributed to J A Asenjo.

At least 19 recordsLinked to original sources

PubMed2006

Mathematical modeling of elution curves for a protein mixture in ion exchange chromatography and for the optimal selection of operational conditions.

Elution curves in ionic exchange chromatography (IEC) for a three-protein mixture (alpha-lactoalbumin, ovalbumin, and beta-lactoglobulin), carried out under different flow rates and ionic strength conditions, were simulated using two different mathematical models. These models were the Plate Model and the more fundamentally based Rate Model. Relatively low protein concentrations were used to avoid protein-protein interactions. Simulated elution curves were compared with experimental data not used for parameter identification. Deviation between experimental data and the simulated curves using the Plate Model was less than 0.0189 (absorbance units); a slightly higher deviation [0.0252 (absorbance units)] was obtained when the Rate Model was used. A cost function was built that included the effect of the different production stages, namely fermentation, purification, and concentration. These considered the effect on the performance of IEC; yield, purity, concentration and the time needed to accomplish the separation. Operational conditions in the IEC such as flow rate, ionic strength gradient and the operational time can be selected using this model in order to find the minimum cost for the protein production process depending on the characteristics of the final product desired such as purity and yield. This cost function was successfully used for the selection of the operational conditions as well as the fraction of the product to be collected (peak cutting) in IEC. It can be used for protein products with different characteristics and qualities, such as purity and yield, by choosing the appropriate parameters.

Chromatography, Ion Exchange↗

PubMed2005

Correlation for the partition behavior of proteins in aqueous two-phase systems: effect of surface hydrophobicity and charge.

Correlations to describe the effect of surface hydrophobicity and charge of proteins with their partition coefficient in aqueous two-phase systems were investigated. Polyethylene glycol (PEG) 4000/phosphate, sulfate, citrate, and dextran systems in the presence of low (0.6% w/w) and high (8.8% w/w) levels of NaCl were selected for a systematic study of 12 proteins. The surface hydrophobicity of the proteins was measured by ammonium sulfate precipitation as the inverse of their solubility. The hydrophobicity values measured correlated well with the partition coefficients, K, obtained in the PEG/salt systems at high concentration of NaCl (r = 0.92-0.93). In PEG/citrate systems the partition coefficient correlated well with protein hydrophobicity at low and high concentrations of NaCl (r = 0.81 and 0.93, respectively). The PEG/citrate system also had a higher hydrophobic resolution than other systems to exploit differences in the protein's hydrophobicity. The surface charge and charge density of the proteins was determined over a range of pH (3-9) by electrophoretic titration curves; PEG/salt systems did not discriminate well between proteins of different charge or charge density. In the absence of NaCl, K decreased slightly with increased positive charge. At high NaCl concentration, K increased as a function of positive charge. This suggested that the PEG-rich top phase became more negative as the concentration of NaCl in the systems increased and, therefore, attracted the positively charged proteins. The effect of charge was more important in PEG/dextran systems at low concentrations of NaCl. In the PEG/dextran systems at lower concentration of NaCl, molecular weight appeared to be the prime determinant of partition, whereas no clear effect of molecular weight could be found in PEG/salt systems.

Complex Mixtures↗

PubMed2005

A theory of protein-resin interaction in hydrophobic interaction chromatography.

Docking simulations were performed in order to investigate surface area of interaction between several ribonucleases and a reduced model for the hydrophobic moiety used in Phenyl Sepharose using the program AutoDock 3.0. For each ribonucelase, 80 independent simulations with populations consisting of 100 random structures were performed and from these the most probable docked protein-ligand conformations were obtained. A new methodology was used to select the most probable conformations, based on qualitative and quantitative considerations. The interacting amino acids in each protein were identified. The average surface hydrophobicity of the interfacial zone (local hydrophobicity, LH) was determined. The LH showed a high correlation level (r2 = 0.99) with the "hydrophobic contact area" (HCA) experimentally determined for the different ribonucleases as well as with the dimensionless retention time (r2 = 0.90). This study allowed us to identify the zones on the protein surface most probably involved in protein retention in HIC, without tedious experimental work. Given the good correlation level obtained, this new methodology may constitute a novel approach that could be used to predict protein behavior in HIC.

Binding Sites↗

PubMed2004

Effect of electrostatic energy on partitioning of proteins in aqueous two-phase systems.

An attempt has been made to adopt a different approach to evaluate the effect of a protein's charge on its partitioning behaviour in PEG/salt aqueous two-phase systems (ATPS). This has been done using a computer methodology (DelPhi) that allows the calculation of the electrostatic solvation energy that charged proteins present in a particular media such as aqueous polymer-salt systems. This calculation was done for the protein in each of the phases and a correlation was investigated that related the electrostatic energy difference of the protein in each of the phases and its partition coefficient in ATPS. Such correlation resulted in a statistical model that also included the effect of molecular weight and a shape factor at each particular pH. A global correlation which included the effect of pH was also found. All the correlations were statistically evaluated and gave good results.

Chromatography, Liquid↗

PubMed2004

Effect of surface hydrophobicity distribution on retention of ribonucleases in hydrophobic interaction chromatography.

The effect of surface hydrophobicity distribution of proteins on retention in hydrophobic interaction chromatography (HIC) was investigated. Average surface hydrophobicity as well as hydrophobic contact area between protein and matrix were estimated using a classical thermodynamic model. The applicability of the model to predict protein retention in HIC was investigated on ribonucleases with similar average surface hydrophobicity but different surface hydrophobicity distribution. It was shown experimentally that surface hydrophobicity distribution could have an important effect on protein retention in HIC. The parameter "hydrophobic contact area," which comes from the thermodynamic model, was able to represent well the protein retention in HIC with salt gradient elution. Location and size of the hydrophobic patches can therefore have an important effect on protein retention in HIC, and the hydrophobic contact area adequately describes this.

Chromatography, Liquid↗

PubMed2003

Study of recombinant micro-organism populations characterized by their plasmid content per cell using a segregated model.

Numerous observations from recombinant systems have shown that properties such as the specific cell growth rate and the plasmid-free cell formation rate are related, not only to the average plasmid content per cell, but also to the plasmid distribution within a population. The plasmid distribution in recombinant cultures can have an effect on the culture productivity that cannot be modelled using average values of the overall culture. The prediction of the behaviour of a plasmid content distribution and its causes and effects can only be studied using segregated models. A segregated model that describes populations of recombinant cells characterized by their plasmid content distribution has been developed. This model includes critical causes of recombinant culture instability such as the plasmid partition mechanism at cell division, plasmid replication kinetics and the effect of the plasmid content on the specific growth rate. The segregated model allows investigation of the effect of each of these causes and that of the plasmid content distribution on the observable behaviour of a recombinant culture. The effect of two partitioning mechanisms (Gaussian distribution and binomial distribution) on culture stability was investigated. The Gaussian distribution is slightly more stable. A small plasmid replication rate constant results in a very unstable culture even after short periods of time. This instability is dramatically improved for a larger value of this constant, hence improving protein synthesis. For a very narrow initial plasmid distribution, a given plasmid replication rate and partitioning mechanism can become broad even after a relatively short period of time. In contrast, a very "broad" initial distribution gave rise to a "Gamma-like" distribution profile. If we compare the results obtained in the simulations of the segregated model with those of the non-segregated one (average model), the latter model predicts much more stable behaviour, thus these average models cannot predict culture instability with the same precision. When compared with the experimental results, the segregated model was able to predict the practical behaviour with accuracy even in a system with a high plasmid content per cell and a high rate of plasmid-free cell formation which could not be achieved with a non-segregated model.

Journal Article↗

PubMed2002

Phase separation rates of aqueous two-phase systems: correlation with system properties.

The kinetics of phase separation in aqueous two-phase systems have been investigated as a function of the physical properties of the system. Two distinct situations for the settling velocities were found, one in which the light, organic-rich (PEG) phase is continuous and the other in which the heavier, salt-rich (phosphate) phase is continuous. The settling rate of a particular system is a crucial parameter for equipment design, and it was studied as a function of measured viscosity and density of each of the phases as well as the interfacial tension between the phases. Interfacial tension increases with increasing tie line length. A correlation that describes the rate of phase separation was investigated. This correlation, which is a function of the system parameters mentioned above, described the behavior of the system successfully. Different values of the parameters in the correlation were fitted for bottom-phase-continuous and top-phase-continuous systems. These parameters showed that density and viscosity play a role in the rate of separation in both top continuous- and bottom continuous-phase regions but are more dominant in the continuous top-phase region. The composition of the two-phase system was characterized by the tie line length. The rate of separation increased with increasing tie line length in both cases but at a faster rate when the bottom (less viscous) phase was the continuous phase. These results show that working in a continuous bottom-phase region is advantageous to ensure fast separation.

Models, Chemical↗

PubMed2001

Process performance models in the optimization of multiproduct protein production plants.

In this work we propose a model that simultaneously optimizes the process variables and the structure of a multiproduct batch plant for the production of recombinant proteins. The complete model includes process performance models for the unit stages and a posynomial representation for the multiproduct batch plant. Although the constant time and size factor models are the most commonly used to model multiproduct batch processes, process performance models describe these time and size factors as functions of the process variables selected for optimization. These process performance models are expressed as algebraic equations obtained from the analytical integration of simplified mass balances and kinetic expressions that describe each unit operation. They are kept as simple as possible while retaining the influence of the process variables selected to optimize the plant. The resulting mixed-integer nonlinear program simultaneously calculates the plant structure (parallel units in or out of phase, and allocation of intermediate storage tanks), the batch plant decision variables (equipment sizes, batch sizes, and operating times of semicontinuous items), and the process decision variables (e.g., final concentration at selected stages, volumetric ratio of phases in the liquid-liquid extraction). A noteworthy feature of the proposed approach is that the mathematical model for the plant is the same as that used in the constant factor model. The process performance models are handled as extra constraints. A plant consisting of eight stages operating in the single product campaign mode (one fermentation, two microfiltrations, two ultrafiltrations, one homogenization, one liquid-liquid extraction, and one chromatography) for producing four different recombinant proteins by the genetically engineered yeast Saccharomyces cerevisiae was modeled and optimized. Using this example, it is shown that the presence of additional degrees of freedom introduced by the process performance models, with respect to a fixed size and time factor model, represents an important development in improving plant design.

Automation↗

PubMed2001

Overproduction, purification, and characterization of beta-1,3-glucanase type II in Escherichia coli.

An Escherichia coli recombinant system produced soluble and full-length beta-1,3-glucanase type II (BglII) cloned from the yeast-lytic actinomycete Oerskovia xanthineolytica. The expression system was designed to produce recombinant BglII with a six-histidine peptide fused to the carboxy end of the protein. The expression level was optimized to produce 30% of total protein of E. coli as the recombinant protein, releasing 75% to the extracellular space. The 43-kDa recombinant protein was purified by IMAC to homogeneity and its molecular and biochemical characteristics were studied, showing that there are no important functional differences with those properties described for the BglII purified from O. xanthineolytica.

Actinomycetales↗

PubMed2001

Isolation, purification and preliminary characterization of cryophilic proteases of marine origin.

The isolation and preliminary characterization of a trypsin-like protease with high activity at 20 degrees C is described. This protease was isolated from Antarctic krill (Euphasia superba) by a two-step chromatography process and the use of zymogram analyses. The protease has a molecular weight of 30 kDa and a pI of 4.1. Its specific activity at 20 degrees C on BAPNA is 0.5 U/mg.

Animals↗

PubMed2000

Model for the partition of metal ions in aqueous two-phase systems.

A model for the partition of metal ions in aqueous two-phase systems (ATPS) has been developed. The partition coefficient of a metal ion D(M), is a function of several variables of the ion (size, charge and electronegativity), characteristics of the ATPS such as type of salt, salt concentration and PEG concentration and additional inorganic salt present in the ATPS. The model has been tested for complex anions of BiX4- (BiCl4-, BiBr4- and BiI4-) and cations from groups I and II (Na+, Cs+, Ca2+, Sr2+ and Ba2+) giving a good correlation in both systems. It was found that for these systems partition coefficient increases with ion size and the variables Y which is a characteristic of the ATPS and Z which is a characteristic of the additional salt present in the system. The partition coefficient of BiX4- increases with the variable X which is a characteristic of the electrical interactions of the metal ion. The cations from groups II and I exhibit the opposite behavior, which is attributable to the ion charge.

Cations↗

PubMed2000

Drowning-out crystallisation of sodium sulphate using aqueous two-phase systems.

A novel method to obtain crystals of pure, anhydrous salt, using aqueous two-phase systems was studied. A concentrated salt solution is mixed with polyethylene glycol (PEG), upon which three phases are formed: salt crystals, a PEG-rich liquid and a salt-rich liquid. After removal of the solid salt, a two-phase system is obtained. Both liquid phases are recycled, allowing the design of a continuous process, which could be exploited industrially. The phase diagram of the system water-Na2SO4-PEG 3350 at 28 degrees C was used. Several process alternatives are proposed and their economic potential is discussed. The process steps needed to produce sodium sulphate crystals include mixing, crystallisation, settling and, optionally, evaporation of water. The yield of sodium sulphate increases dramatically if an evaporation step is used.

Crystallization↗

PubMed1999

Human colon adenocarcinomas express a MUC1-associated novel carbohydrate epitope on core mucin glycans defined by a monoclonal antibody (A10) raised against murine Ehrlich tumor cells.

A monoclonal antibody (mAb; A10) raised against murine Ehrlich tumor cell surface carbohydrates was tested for reactivity with human normal and malignant tissues. A10 reacted strongly, with a high proportion of adenocarcinomas arising from colon and other tissues but not with breast carcinomas or other malignant tumors. Normal tissues were virtually A10 unreactive, except for the duct cells from breast and pancreas and some bronchial mucosae. Ultrastructural studies showed mAb A10 immunolabeling of both microvilli and mucin droplets in colon cancer cells but not in normal absorptive or globet cells. A10 reacted strongly with mucin-enriched fractions from colon cancer tissues and HT-29 xenografts but not from normal colon tissues. A10 epitope was carried on MUC1 derived from colon adenocarcinomas and probably on other mucin species, although not on MUC2 molecules. A10 epitope was resistant to exoglycosidases and periodate oxidation but sensitive to the Smith's degradation and beta-elimination, suggesting the involvement of O-linked carbohydrates in nonterminal reducing positions. A mucin-type glycosidic linkage was supported because of the lack of A10 reactivity with HT-29 cells grown with phenyl-N-acetyl-alpha-D-galactosaminide. Deglycosylation studies with trifluoromethanesulfonic acid pointed to the involvement of core mucin glycans in the A10 epitope. This epitope was resistant to protease, O- and N-glycanase treatments carried out on trifluoromethanesulfonic acid-deglycosylated mucins. Inhibition studies with core 1, core 2, core 3, and core 6 suggested the latter [GlcNAcbeta(1-6)GalNAc] as being involved in A10 epitope. Taken together, the present results point to A10 defining a core 6-related epitope on core mucin glycans expressed by colon cancer MUC1 not previously associated with human cancer.

Adenocarcinoma↗

PubMed1999

LOH at the APC/MCC gene (5Q21) is frequent in early stages of non-small cell lung cancer.

Lung cancer is the leading cause of death in both women and men in the United States and many European countries. Molecular cytogenetic and LOH analyses of non-small cell lung cancer have shown somatic genetic alterations in a variety of chromosomes, such as 1p, 3p, 5q, 8p, 9p, 11p, 11q and 17p. Allelic deletions of the known tumor suppressor gene APC at 5q21 are frequently observed in advanced stages of lung cancer and have been correlated with poor prognosis in previous reports. We investigated 33 cases of NSCL for LOH at 5q21: 22 squamous cell and 11 adenocarcinomas. Normal and tumor cells were microdissected from paraffin embedded tissues and PCR amplification was performed utilising the specific markers D5S299 and D5S346 at 5q21 and PYGM at 11q13, respectively. Clinicopathological data, survival and recurrence rates were obtained in all cases. We detected LOH at 5q21 in 4/9 (44%) informative adenocarcinomas and in 13/16 (81%) informative SCC. LOH was frequent in early stages (12/15 stage I cases) and did not correlate with recurrence or poor survival. Our results show that LOH at 5q21 is more frequent in squamous cell carcinomas than in adenocarcinomas, is frequent in early stages of the disease, and does not have prognostic significance.

Adenocarcinoma↗

PubMed1999

Flow cytometric DNA ploidy analysis of oral cancer comparison with histologic grading.

It has been reported that DNA content analysis provides prognostic information eliminating the subjective component involved in routine microscopic decision making. In an attempt to establish a relationship between pathological findings and flow cytometric analysis, 36 formalin-fixed, paraffin-embedded tumour tissue samples were prepared according to Hedley's method and analysed by means of an Epics Profile II flow cytometer. DNA aneuploidy was observed in 15 tumours (41%). A statistically significant correlation was identified between DNA index and mitoses, cellular response and degree of differentiation, but not the ploidy status. It was not possible to identify a significant association of sex, age, and site of the tumour to DNA index. We found a strong correlation between histologic malignancy and DNA index; an increase in DNA index as malignancy score increases was noted. It was concluded that DNA index shows a good correlation with the histologic features of oral cancer, being a complement of differentiation and histologic grading analysis. The use of DNA analysis as a complement to pathological studies would help to diminish the subjective component of assessment of head and neck cancers. Ploidy status was not statistically associated with the differentiation of tumours.

Adult↗

PubMed1998

Aqueous two-phase systems for protein separation. Studies on phase inversion.

Phase equilibrium studies were done with the PEG 400-phosphate system, obtaining equilibrium binodal lines, tie lines and phase inversion points. A method of calculation of the critical point on the binodal curve is described. The influence of the presence of NaCl in solution was studied, and the comparative results are presented. It was found that in some range of concentration the shift produced in the binodal line can be important. The rate of phase separation can be used as an indication of which of the phases is continuous. Using this method the phase inversion point can be determined in the system for each tie line. A range of ambiguity was found, where the continuity of the phases is affected not only by the composition of the mixture, but also by the fluid dynamics. Within this range, gentle agitation produces a bottom-continuous suspension. while strong agitation produces a top-continuous suspension. Two inversion points exist therefore on each tie line, delimiting on the phase equilibrium plane a region where the phase continuity depends on fluid dynamics. The convergence of this region towards the critical point can be used to control of the consistency of the experimental data.

Chemistry Techniques, Analytical↗

PubMed1998

On the kinetics of phase separation in aqueous two-phase systems.

The effect of the tie-line location (phase volume ratio) on the kinetics of phase separation in batch PEG/salt aqueous two-phase systems (ATPS) has been investigated. PEG/sulphate systems with a stability ratio (sr) of 0.34 and 0.37 and relative tie-line lengths in the range 0.1 to 0.6 for a continuous top phase and in the range 0.03 to 0.15 for a continuous bottom phase were used in the batch studies. A continuous settler was designed with three different inlet geometries. Phase separation is much faster when the bottom phase is continuous and in this case the location on the tie-line and the presence or absence of Bacillus subtilis extract makes little difference. When the top phase is continuous the relative sizes of the phases (phase ratio, R. relative distance on tie-line, rd) has an important effect, the larger the top phase (larger R and rd) the slower the phase separation. The presence of Bacillus extract also makes the operation slower which is more marked at the largest values of R (and rd). At the largest volume ratios (R or rd) three different settling regions have been recognised, a region of coalescence, a region of drops moving to the interphase and a region where drops queue at the interphase to coalesce into the large phase. A modified correlation that takes into account the location on the tie-line and thus volume ratio (R) and relative distance (rd) has been proposed and successfully tested. The behavior of batch and continuous systems in the presence and absence of Bacillus subtilis extract in systems with continuous bottom phase was also studied. The settling velocity was lower in the continuous than in the batch systems, and in both cases the initial rate was lower in the presence of Bacillus extract.

Bacillus subtilis↗

PubMed1998

Flow cytometric analysis of nuclear DNA content in oral leukoplakia.

DNA ploidy has been studied in 31 paraffin-embedded specimens of oral leukoplakia and in 10 specimens of oral leukoplakia that have undergone malignant transformation after a 5-year observation period. Three out of 31 specimens (9.7%) showed an aneuploid DNA pattern, with DNA indices of 1.17, 1.28 and 1.32. It was not possible to establish a statistically significant difference of DNA ploidy related to the presence or absence of dysplasia. One out of the 10 oral leukoplakias that underwent malignant transformations had a multiploid pattern with DNA indices of 1.44 and 2.37. In this study the DNA index has not proved to be of value in the identification of dysplastic leukoplakia among the non-dysplastic ones.

Aneuploidy↗