Biomedical subjects

B A Andrews

Publications and source records attributed to B A Andrews.

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↗

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↗

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

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↗

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↗

PubMed1996

Protein partitioning equilibrium between the aqueous poly(ethylene glycol) and salt phases and the solid protein phase in poly(ethylene glycol)-salt two-phase systems.

True partitioning behaviour, which is independent of the protein concentration in aqueous two-phase systems, only occurs at relatively low protein concentration. The actual concentration limit depends on the properties of the protein. When the concentration of a protein exceeds relatively low values, precipitation at the interface can be observed. This protein precipitate is in equilibrium with the protein solubilized in each of the phases. This paper discusses the effect of protein solubility in view of the equilibrium of the protein concentration between the aqueous poly(ethylene glycol) and salt phases and the solid protein phase using three proteins. It was found that only rarely will the proteins be completely in solution as the concentration is increased until a solubility limit is reached and then the protein precipitates fully out of solution. A behaviour that came close to this was only seen in one case out of six. In virtually all cases, a third phase is formed which represents a solid aggregate phase which is in equilibrium with the other two, largely aqueous, phases. As the overall concentration of protein in the system is increased and the concentration in the top and bottom aqueous phases increases, the pseudo concentration in the solid-phase, C's, also increases. This could have interesting implications in terms of the amount of water associated with this phase and it certainly means that in this particular case, the solid phase is not a crystal.

Aspergillus niger↗

PubMed1996

Partitioning and purification of monoclonal antibodies in aqueous two-phase systems.

The partition behaviour of pure IgG in aqueous two-phase systems was examined in order to investigate the effects of changes in phase properties on the partition coefficient, K. Factors such as molecular weight (M.W.) of PEG, pH, and concentration of NaCl and other salts were all found to influence K. Due to the high level of interaction between the phase components, optimal conditions were found using a factorial design. Using this methodology it was possible to find conditions which gave extremely high values of K for the IgGs (> 100). Partition behaviour of some potential contaminants, BSA and transferrin, were also studied. All the components were characterized for hydrophobicity, pI and M.W. Conditions were chosen under which the partition coefficients of the contaminants was low (pH 6, 15% PEG 1450, 14% Phosphate and 12% NaCl). Precipitation of IgG was also minimized. These conditions were chosen for the extraction of IgG from the contaminants in a crude concentrated supernatant. The presence of culture media contaminants had a strong effect in the systems and hence on the partition of IgG which was lower than for pure IgG. The ratio of KIgG/Kcont was > 25. The conditions chosen for the first forward extraction into the PEG rich phase were 15% PEG 1450, 14% phosphate 12% NaCl and pH 5.5. Virtually all IgG partitioned to the top phase and the contaminants to the bottom phase. Optimal conditions for the back extraction of the IgGs into a bottom salt phase were also found (addition of 14% phosphate with no NaCl). An industrial serum free, crude, concentrated culture supernatant of hybridoma produced murine IgG, with a relatively low level of protein contaminants (14% IgG purity) was processed in this system. After the back extraction the contaminants were reduced 18 fold giving IgG with 80% purity. A 5.9 fold purification was obtained out of a 7.3 maximum possible (at 100% pure IgG). All of the IgG was recovered.

Antibodies, Monoclonal↗

PubMed1995

Purification of virus like particles from yeast cells using aqueous two-phase systems.

Aqueous two-phase systems have been used for the separation and purification of recombinant virus-like particles (VLPs) from a yeast cell homogenate. Systems have been evaluated on the basis of their ability to separate VLPs from cell debris and from contaminating proteins. Two strategies are proposed for VLP separation, both involve the use of two PEG/salt aqueous two-phase systems. For separation of VLPs from cell debris systems composed of PEG 400 or 600 and (NH4)2SO4 or PEG 4000/MgSO4 can be used and for separation of VLPs from proteins PEG 4000/(NH4)2SO4 or MgSO4 and Na2SO4 are effective. The molecular weight of the PEG used and the use of additional salts (e.g. NaCl) greatly influence the effectiveness of the systems.

Ammonium Sulfate↗

PubMed1994

Role of early postoperative surface echocardiography in the pediatric cardiac intensive care unit.

OBJECTIVE: To compare surface echocardiographic data with catheterization and surgical observation as a way of deciding on the need to reoperate to correct hemodynamically important sequelae following pediatric cardiac surgery; to determine the false-negative diagnosis rate of surface echocardiography. DESIGN: Case series. SETTING: Tertiary-care center, pediatric cardiac intensive care unit. PATIENTS: All 39 patients who underwent reoperation because of hemodynamically significant anatomic sequelae following primary or elective secondary surgery in 1 calendar year. INTERVENTIONS: None. MEASUREMENTS: Two-dimensional and color Doppler ultrasound assessment of anatomy and physiology following cardiac surgery. RESULTS: In 85 percent, surface echocardiography provided sufficient information for surgeons to reoperate on the same admission. Detection of important residual shunts or arterial stenoses and identification of anatomic causes of pulmonary undercirculation (or overcirculation) in palliated single ventricle are feasible. CONCLUSION: Early postoperative surface echocardiography is a viable way to decide on the hemodynamic adequacy of cardiac surgery.

Adolescent↗

PubMed1993

Changes in ventricular geometry early after Fontan operation.

Acute removal of a ventricular volume overload, in the face of relatively unchanging ventricular mass, results in geometric alterations that can impair diastolic ventricular performance after Fontan operation. We investigated whether geometric alterations were (1) more severe after Fontan operation than after hemi-Fontan operation, (2) more severe in infants than in children, and (3) more severe in the morphologic right ventricle than in the morphologic left ventricle. We studied 22 patients, 11 with hypoplastic left heart syndrome and 11 with a functionally single morphologic left ventricle. After Fontan operation, right ventricular end-diastolic volume declined by 52% from 36.6 +/- 12.2 to 17.5 +/- 9.3 mL. Right ventricular wall thickness increased from 6.8 +/- 1.3 to 7.5 +/- 1.8 mm. Left ventricular end-diastolic volume diminished by 40% from 57.6 +/- 23.3 to 34.8 +/- 17.6 mL. Left ventricular wall thickness increased from 7.0 +/- 2.1 to 8.7 +/- 1.9 mm. The alterations in right ventricular geometry are more marked early after Fontan operation than early after the hemi-Fontan procedure. Geometric alterations in the left ventricle appear to be more severe in infants than in children. Finally, alterations in the morphologic right ventricle appear to be of similar magnitude to those in the morphologic left ventricle.

Body Surface Area↗

PubMed1993

Selective release of recombinant protein particles (VLPs) from yeast using a pure lytic glucanase enzyme.

We have used a pure lytic glucanase enzyme to selectively release recombinant 60 nm protein particles (virus like particles or VLPs) from yeast cells. Although the protease components of the lytic enzyme complexes were found to degrade the VLPs, purified glucanase enzymes from these complexes (derived from Cytophaga sp. and Oerskovia sp.) produced cell lysis without degradation and released the VLPs in the absence of mercaptoethanol, a reducing agent commonly used in cell lysis. The Oerskovia glucanase enzyme released the recombinant protein particles selectively as it only produced ca. 17% cell lysis compared to the use of the total lytic enzyme preparation. The use of osmotic supports did not improve the recovery of VLPs, however, treatment of the enzymatically lysed pellet with Triton X-100 did increase the amount of VLPs released. This 'selectivity', which results in the release of the recombinant particles with only a fraction of contaminating proteins, represents an improvement over presently used mechanical or enzymatic cell disruption processes.

Actinomycetales↗

PubMed1991

Rational design of purification processes for recombinant proteins.

Recent developments in the rational design of purification processes for recombinant proteins are discussed. A review of the main issues involved in process design for protein separation and purification is presented with particular emphasis on the challenges posed by recombinant proteins. This includes physicochemical characterization of target protein and main contaminants, the use of rigorous mathematical modeling and process simulation as well as the development of an expert system and the application of this technology for optimization and design of large scale processes. An expert system for selection of optimal protein separation sequences will give the user a number of alternatives chosen on the basis of extensive data back-up on proteins and unit operations.

Biotechnology↗

PubMed1990

Ultrasonographic display of complex vascular rings.

Ultrasonographic display of vascular ring anatomy has been limited to single-plane views. This does not readily allow for a three-dimensional interpretation of structural relations. A method that included a sweep consisting of multiple contiguous frontal planes was used in 12 patients with a vascular ring before repair for the evaluation of the arch sidedness and number, brachiocephalic vessel pattern, upper descending thoracic aorta sidedness, ductus arteriosus site or sites and proximal pulmonary arteries; 7 patients had Doppler color flow imaging. Complete imaging of the luminal vascular components was possible in all but one patient. In four other patients, atretic segments of the vascular ring could not be displayed. The addition of Doppler color flow imaging especially aided in the tracing of multiple vascular structures in complex cases and in assessing ductus arteriosus and arch patency. The use of a suprasternal frontal sweep with posterior angulation could display encirclement of the air-filled trachea. Vascular ring segments without lumens could not be displayed.

Aorta, Thoracic↗