Mechanism and kinetics of protein transport in chromatographic media studied by confocal laser scanning microscopy. Part I. The interplay of sorbent structure and fluid phase conditions

An experimental study on the interplay of sorbent structure and fluid phase conditions (pH) has been carried out examining adsorption and transport of bovine serum albumin (BSA) and a monoclonal antibody (IgG 2a) on SP Sepharose Fast Flow and SP Sepharose XL. SP Sepharose Fast Flow is characterised by a relatively open pore network, while SP Sepharose XL is a composite structure with ligand-carrying dextran chains filling the pore space. Both adsorbents have similar ionic capacity. Protein transport and adsorption profiles were evaluated using confocal laser scanning microscopy. Under all investigated conditions, BSA uptake could be adequately explained by a pore diffusion mechanism. The adsorption profiles obtained for IgG 2a, however, indicated that changes in fluid phase conditions as well as a change in the solid phase structure could result in a more complex uptake mechanism as compared to pore diffusion alone. This mechanism results in a fast transport of proteins into the adsorbent, followed by an overshoot of protein in the center of the sorbent and a setback towards a homogeneous adsorption profile.     

Lattice Boltzmann methods for shallow water flow applications

We apply the lattice Boltzmann (LB) method for solving the shallow water equations with source terms such as the bed slope and bed friction. Our aim is to use a simple and accurate representation of the source terms in order to simulate practical shallow water flows without relying on upwind discretization or Riemann problem solvers. We validate the algorithm on problems where analytical solutions are available. The numerical results are in good agreement with analytical solutions. Furthermore, we test the method on a practical problem by simulating mean flow in the Strait of Gibraltar. The main focus is to examine the performance of the LB method for complex geometries with irregular bathymetry. The results demonstrate its ability to capture the main flow features. Copyright © 2007 John Wiley & Sons, Ltd.     

Relaxation of spin polarized 3He by magnetized ferromagnetic contaminants

In the first in a series of three papers on wall relaxation of spin polarized ³He we have reported on a breakdown of relaxation times which is observed after exposing the ³He containing glass cells to a strong magnetizing field. In this third paper we give a quantitative analysis of this phenomenon, based on magnetic signal detection by means of SQUIDs, on the pressure dependence of relaxation times in magnetized cells, as well as on Monte Carlo simulations of ³He-relaxation in a macroscopic dipole field. Our analysis allows to identify the contaminants as being aggregates of dust-like Fe₃O₄ particles (magnetite) with a radius R ? 10 mR \approx 10~\mu m and a remanent magnetic moment of the order of m ≈O(10 ^-10 ^{-10}~ A m²). The particles are located at or close to the inner glass surface.     

A lattice Boltzmann method for immiscible multiphase flow simulations using the level set method

We consider the lattice Boltzmann method for immiscible multiphase flow simulations. Classical lattice Boltzmann methods for this problem, e.g. the colour gradient method or the free energy approach, can only be applied when density and viscosity ratios are small. Moreover, they use additional fields defined on the whole domain to describe the different phases and model phase separation by special interactions at each node. In contrast, our approach simulates the flow using a single field and separates the fluid phases by a free moving interface. The scheme is based on the lattice Boltzmann method and uses the level set method to compute the evolution of the interface. To couple the fluid phases, we develop new boundary conditions which realise the macroscopic jump conditions at the interface and incorporate surface tension in the lattice Boltzmann framework. Various simulations are presented to validate the numerical scheme, e.g. two-phase channel flows, the Young–Laplace law for a bubble and viscous fingering in a Hele-Shaw cell. The results show that the method is feasible over a wide range of density and viscosity differences.     

Optimal boundary control of glass cooling processes

In this paper, an optimal control problem for glass cooling processes is studied. We model glass cooling using the SP1 approximations to the radiative heat transfer equations. The control variable is the temperature at the boundary of the domain. This results in a boundary control problem for a parabolic/elliptic system which is treated by a constrained optimization approach. We consider several cost functionals of tracking‐type and formally derive the first‐order optimality system. Several numerical methods based on the adjoint variables are investigated. We present results of numerical simulations illustrating the feasibility and performance of the different approaches. Copyright © 2004 John Wiley & Sons, Ltd.     

(S,S)-2,3-Dihydroxy-2,3-dihydrobenzoic acid: microbial access with engineered cells of Escherichia coli and application as starting material in natural-product synthesis

Cyclohexadiene-trans-5,6-diols such as (S,S)-2,3-dihydroxy-2,3-dihydrobenzoic acid (2,3-trans-CHD) have been shown to be of importance as chiral starting materials for the syntheses of bioactive substances, especially for the syntheses of carbasugars. By using methods of metabolic-pathway engineering, the Escherichia coli genes entB and entC, which encode isochorismatase and isochorismate synthase, were cloned and over-expressed in E. coli strains with a deficiency of entA, which encodes 2,3-dihydroxybenzoate synthase. A 30-fold increase in the corresponding EntB/EntC enzyme activities affects the accumulation of 2,3-trans-CHD in the cultivation medium. Although the strains did not contain deletions in chorismate-utilising pathways towards aromatic amino acids, neither chorismate nor any other metabolic intermediates were found as by-products. Fermentation of these strains in a 30 L pH-controlled stirred tank reactor showed that 2,3-trans-CHD could be obtained in concentrations of up to 4.6 g L(-1). This demonstrates that post-chorismate metabolites are accessible on a preparative scale by using techniques of metabolic-pathway engineering. Isolation and separation from fermentation salts could be performed economically in one step through anion-exchange chromatography or, alternatively, by reactive extraction. Starting from 2,3-trans-CHD as an example, we established short syntheses towards new carbasugar derivatives.     

Mechanism and kinetics of protein transport in chromatographic media studied by confocal laser scanning microscopy. Part II. Impact on chromatographic separations

The impact of different transport mechanism on chromatographic performance was studied by confocal laser scanning microscopy (CLSM) for solutions containing bovine serum albumin (BSA) and monoclonal IgG 2a under different solid- and fluid-phase conditions. During this investigation, a clear influence of the uptake mechanism on the affinity of the respective proteins for the different adsorbents and thus separation performance of the chromatographic process could be observed. For the system SP Sepharose Fast Flow at pH 4.5 pore diffusion could be ascribed to be the dominant transport mechanism for both proteins and the adsorption profiles resembled a pattern similar to that described by the 'shrinking core' model. Under these conditions a significantly higher affinity towards the adsorbent was found for BSA when compared to IgG 2a. With changing fluid- and solid-phase conditions, however, a change of the transport mode for IgG 2a could be detected. While the exact mechanism is still unresolved it could be concluded that both occurrence and magnitude of the now governing transport mechanism depended on protein properties and interaction with the adsorbent surface. For the system SP Sepharose XL at pH 5.0 both parameters leading to the change in IgG 2a uptake were combined resulting in a clear change of the system affinity towards the IgG 2a molecule, while BSA adsorption was restricted to the most outer shell of the sorbent.     

Lattice Boltzmann Simulation of Free-Surface Temperature Dispersion in Shallow Water Flows

We develop a lattice Boltzmann method for modeling free-surface temperature dispersion in the shallow water flows. The governing equations are derived from the incompressible Navier-Stokes equations with assumptions of shallow water flows including bed frictions, eddy viscosity, wind shear stresses and Coriolis forces. The thermal effects are incorporated in the momentum equation by using a Boussinesq approximation. The dispersion of free-surface temperature is modelled by an advection-diffusion equation. Two distribution functions are used in the lattice Boltzmann method to recover the flow and temperature variables using the same lattice structure. Neither upwind discretization procedures nor Riemann problem solvers are needed in discretizing the shallow water equations. In addition, the source terms are straightforwardly included in the model without relying on well-balanced techniques to treat flux gradients and source terms. We validate the model for a class of problems with known analytical solutions and we also present numerical results for sea-surface temperature distribution in the Strait of Gibraltar.     

Effect of phenyl sepharose ligand density on protein monomer/aggregate purification and separation using hydrophobic interaction chromatography

In the large-scale manufacturing and purification of protein therapeutics, multiple chromatography adsorbent lots are often required due to limited absorbent batch sizes or during replacement at the end of the useful column lifetime. Variability in the adsorbent performance from lot to lot should be minimal in order to ensure that consistent product purity and product quality attributes are achieved when a different lot or lot mixture is implemented in the process. Vendors of chromatographic adsorbents will often provide release specifications, which may possess a narrow range of acceptable values. Despite relatively narrow release specifications, the performance of the adsorbent in a given purification process could still vary from lot to lot. In this case, an alternative use test (one that properly captures the lot to lot variability) may be required to determine an acceptable range of variability for a specific process. In this work, we describe the separation of therapeutic protein monomer and aggregate species using hydrophobic interaction chromatography, which is potentially sensitive to adsorbent lot variability. An alternative use test is formulated, which can be used to rapidly screen different adsorbent lots prior to implementation in a large-scale manufacturing process. In addition, the underlying mechanism responsible for the adsorbent lot variability, which was based upon differences in protein adsorption characteristics, was also investigated using both experimental and modeling approaches.     

Modeling the effects of column packing quality and residence time changes on protein monomer/aggregate separation

The packing quality of chromatography columns used for the purification of protein therapeutics is routinely monitored to ensure consistent and reproducible performance. In this work, we used established chromatography models to determine the effect of column packing quality and fluid residence time on the separation of protein therapeutic monomer and aggregate species using a hydrophobic interaction chromatography adsorbent (Phenyl Sepharose Fast Flow). The relationship between the number of theoretical plates, fluid residence time, and column separation performance was quantified using modeling simulations. The simulations showed the separation depended on both the fluid residence time and the number of theoretical plates. However, when the number of theoretical plates was increased to ≥150, the simulations predicted that the separation performance of the column was not significantly improved. The approach described here could be used as a method to quantify acceptable height equivalent of a theoretical plate values for columns, and serve as a tool to understand how column packing quality impacts a given chromatographic separation prior to column scale-up, as well as during the monitoring of column lifetime in the manufacturing of large scale protein therapeutics.