Biomedical subjects

Jörg Thömmes

Publications and source records attributed to Jörg Thömmes.

9 recordsLinked to original sources

PubMed2006

Expanded bed adsorption as a primary recovery step for the isolation of the insulin precursor MI3 process development and scale up.

Expanded bed adsorption (EBA) was evaluated for the isolation of the human insulin precursor MI3, expressed and secreted by the yeast Saccharomyces cerevisiae. The isoelectric point of the insulin precursor (pH 5.3) makes cation exchange a prime candidate for direct adsorption. In order to find a suitable window of operation for the process the adsorption equilibrium was analysed in a wide range of operating conditions (pH and conductivity) and for three different stationary phases. The same array of operating conditions was examined with regard to stable fluidisation of the adsorbents in S. cerevisiae suspensions. Interactions of the yeast with the fluidised stationary phase were investigated by a pulse response technique and the hydrodynamics of the fluidised bed under process conditions by residence time distribution analysis. The case study demonstrates that by parallel examination of product binding and fluidisation quality a window of operation can be found. Analysis of the binding kinetics by breakthrough experiments and modelling led to the definition of a set of operating conditions, which yield a compromise between optimal use of the equilibrium capacity provided by the adsorbent and high throughput required for an industrial separation. After initial experiments on the bench scale the protocol was transferred successfully to pilot scale demonstrating the design of a reliable operation.

Adsorption↗

PubMed2005

Biochemical engineering aspects of expanded bed adsorption.

The economic feasibility of a biotechnological production process is directly linked to the number and efficiency of the processes required during purification in order to reach the requested product specifications. Expanded bed adsorption is an integrated process that combines solid-liquid separation and product recovery into a single unit operation. This approach suggests an increased overall yield, lower requirements for capital investments and consumables and most importantly a reduced process time. The success of an integrative process is, however, closely linked to a detailed understanding of the biochemical principles involved and the constraints arising from feedstock complexity. This paper reviews the hydrodynamic principles that expanded bed adsorption is based on, discusses current developments in resin and column design, and finally presents a methodology for targeted process development in expanded bed adsorption.

Adsorption↗

PubMed2004

The influence of biomass on the hydrodynamic behavior and stability of expanded beds.

Expanded bed adsorption is an innovative chromatographic technology that allows the introduction of particle-containing feedstock without the risk of blocking the bed. Provided a perfectly classified fluidized bed (termed expanded bed) is formed in the crude feedstock and the biomass is not influencing protein transport towards the adsorbent surface, a sorption performance comparable to packed beds is found. The influence of biomass on the hydrodynamic stability of expanded beds is essential and was investigated systematically in this article. Residence-time distribution analyses were performed using model systems and a yeast suspension under various fluid-phase conditions. It is demonstrated that three factors (biomass/adsorbent interactions, biomass concentration, and flow rate) play an interdependent role disturbing the classified fluidization of an expanded bed. A clear correlation between the degree of aggregative fluidization--obtained by PDE modeling of RTD data--and the expansion behavior of the fluidized bed has been found. Thus, combining three analytical methods, namely cell transmission index analysis, expansion analysis, and RTD analysis provides a solid base for understanding and control of the fluidization behavior and thus further process design during the initial phase of process development.

Algorithms↗

PubMed2003

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.

Chromatography, Ion Exchange↗

PubMed2003

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.

Adsorption↗

PubMed2003

(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.

Benzoates↗

PubMed2003

Biomass/adsorbent electrostatic interactions in expanded bed adsorption: a zeta potential study.

Expanded bed adsorption is an integrated technology that allows the introduction of particle-containing feedstock without the risk of blocking the bed. The biomass particles contained in the feedstock have to be treated as an integral part of the process and potential interactions between suspended biomass and the adsorbent must be excluded during process design. Because the electrostatic forces dominate the interactions between the biomass and adsorbent, the zeta potential has been studied as a tool to characterize biomass/adsorbent electrostatic interactions. The zeta potentials of four types of biomass (yeast intact cells, yeast homogenate, Escherichia coli intact cells, and E. coli homogenate) and two types of ion exchanger were measured systematically at varying process conditions. Using the cell transmission index from biomass pulse-response experiments as a parameter, the relations between zeta potential and the biomass/adsorbent interaction were evaluated. Combining the influences from zeta potential of adsorbent (zeta(a)), zeta potential of biomass (zeta(b)), and biomass size (d), parameter (-zeta(a)zeta(b)d) was found to be an appropriate indicator of the biomass/adsorbent interactions in expanded beds under various liquid-phase conditions for different types of biomass. The threshold value of parameter (-zeta(a)zeta(b)d) can be defined as 120 mV(2) microm for cell transmission of >90%, which means that systems with (-zeta(a)zeta(b)d) < 120 may have a considerable probability of forming stable expanded beds in a biomass suspension under the particular experimental conditions.

Adsorption↗

PubMed2003

Stability of expanded beds during the application of crude feedstock.

Expanded bed adsorption is an integrated technology that allows the introduction of a particle containing feedstock without the risk of blocking the bed. Provided a perfectly classified fluidized bed (termed expanded bed) is formed in the crude feed, a sorption performance comparable to packed beds is found. During the application of biomass containing samples to stable expanded beds an increase in bed expansion due to the higher density and viscosity of the feed is encountered. In this article it is investigated whether the expanded bed condition is also fulfilled during the transition in bed expansion from lower to higher density (i.e., from an equilibration buffer to a biomass containing feedstock). Residence time distribution analyses were performed by using model systems and a yeast suspension during this transition phase. It is shown that in systems in which the biomass does not interact with the fluidized stationary phase, the perfectly classified fluidization is maintained also during this transition phase regardless of the type of feedstock. Additional bed expansion takes place in an "ordered" manner without compromising bed stability. In case of biomass/adsorbent interactions, a deterioration in bed stability is found directly when the crude feed is loaded.

Biomass↗

PubMed2002

Dynamics of protein uptake within the adsorbent particle during packed bed chromatography.

A new experimental set-up for on-line visualization of the intra-particle uptake kinetics during packed bed chromatography has been designed and tested. Confocal laser scanning microscopy was used to analyze the dynamics of protein adsorption to porous stationary phases. In combination with this, a flow cell was developed that could be packed with chromatography media and operated as a fully functional mini-scale chromatography column. Adsorption profiles of single- and two-component mixtures containing BSA and IgG 2a during packed bed cation-exchange chromatography were investigated. The two proteins appear to exhibit different transport characteristics. For BSA a classical "shrinking core" behavior could be detected. The profiles obtained during IgG 2a adsorption point toward a different transport mode, which deviates from the classical pore-diffusion picture. For the two-component system, a superposition of the single-component profiles combined with a classical displacement of the weaker bound protein species was found. The results indicate that depending on the adsorbed protein the uptake can vary tremendously, even for adsorption to the same chromatographic support. It is clearly shown that the new microcolumn allows in situ quantitative investigations of protein adsorption dynamics within a single particle, which adds a new tool to the available methods for characterizing and optimizing protein adsorption chromatography.

Adsorption↗