Biomedical subjects

Rainer Hahn

Publications and source records attributed to Rainer Hahn.

15 recordsLinked to original sources

PubMed2007

Mapping of Malus domestica allergens by 2-D electrophoresis and IgE-reactivity.

The importance of apple allergens has been repeatedly emphasized, and their presence has been confirmed both in pollen and in fruits. In the present study, a combination of proteomic tools have been used to build a complete allergen map of apple. The water-soluble fraction of an apple extract was precipitated using a phenol-based procedure and separated by 2-DE. Initially four previously classified allergens, Mal d 1, Mal d 2, Mal d 3 and Mal d 4, could be identified in Western blots with polyclonal rabbit antibodies directed to the four respective allergens, and subsequently matched to the bands recognized by several patient sera. Further, all four known apple allergens were localized on a 2-DE map and they were matched with spots recognized by sera of patients with different allergic patterns. Moreover, a new, putative allergen could be identified using MS. We evaluated the influence of post-translational modifications and the immunoreactivity under different analytical conditions. The comparison of different visualization methods for 2-DE gels and blots revealed that even very low concentrations of the intact epitopes are detectable by IgEs of patients, and therefore might be sufficient to trigger allergic symptoms in sensitized individuals.

Allergens↗

PubMed2006

Hydrophobic interaction chromatography of proteins IV. Kinetics of protein spreading.

Adsorption of proteins on surfaces of hydrophobic interaction chromatography media is at least a two-stage process. Application of pure protein pulses (bovine serum albumin and beta-lactoglobulin) to hydrophobic interaction chromatography media yielded two chromatographic peaks at low salt concentrations. At these salt concentrations, the adsorption process is affected by a second reaction, which can be interpreted as protein spreading or partial unfolding of the protein. The kinetic constants of the spreading reaction were derived from pulse response experiments at different residence times and varying concentrations by applying a modified adsorption model considering conformational changes. The obtained parameters were used to calculate uptake and breakthrough curves for spreading proteins. Although these parameters were determined at low saturation of the column, predictions of overloaded situations could match the experimental runs satisfactorily. Our findings suggest that proteins which are sensitive to conformational changes should be loaded at high salt concentrations in order to accelerate the adsorption reaction and to obtain steeper breakthrough curves.

Adsorption↗

PubMed2006

Dispersion effects in preparative polymethacrylate monoliths operated in radial-flow columns.

Monolithic media have found widespread use as excellent tools for fast analytical separations of small molecules, proteins, pDNA and viruses. Polymethacrylate monoliths with large channels are attractive for capturing large molecules, like immunoglobulins, DNA, and viruses. For preparative purposes, these monoliths are operated in radial flow mode. Band spreading in monoliths is extremely low and mostly dominated by the contribution of extra column effects. The model used here had a single axial dispersion coefficient which lumps together extra column effects and the intrinsic band spreading of the monolithic material to characterize the adsorption of proteins and pDNA on polymethacrylate ion-exchange monoliths. Due to the fact that the performance of the monolith was unaffected by the velocity within the applied range, and due to highly favourable adsorption isotherms, a constant pattern model could be applied to predict preparative runs on radial flow units assuming axial flow for modelling.

Adsorption↗

PubMed2005

Comparison of protein A affinity sorbents III. Life time study.

Protein A affinity chromatography is a popular purification method for immunoglobulins applied at various scales, ranging from micro-tube up to 1000l column format. Three novel high capacity protein A affinity chromatography media have been subjected to a lifetime study using 50 consecutive purification cycles of a cell culture supernatant (CCS) containing a monoclonal antibody. Chromatographic conditions followed protocols used in industrial antibody processing, including stripping and cleaning-in-place of the resins. For all three media, no significant loss of purification performance (measured by sodium dodecylsulfate polyacrylamide gel electrophoresis and analytical size-exclusion chromatography (SEC)) could be observed over 50 cycles. Eluate samples were analyzed for leaked protein A and host cell protein (HCP) content. MabSelect SuRe, the first protein A affinity medium compatible with alkaline regeneration conditions, exhibited the lowest leakage levels, in the range of 1-3 ppm. For the media MabSelect Xtra and ProSep-vA Ultra, leakage levels were in the range of 30-40 ppm. Host cell protein content of eluates from MabSelect Xtra and SuRe were between 300 and 700 ppm, whereas for ProSep-vA Ultra 3000-4000 ppm was achieved.

Cell Line↗

PubMed2005

Comparison of protein A affinity sorbents II. Mass transfer properties.

Protein A affinity chromatography is the standard purification method for isolation of therapeutic antibodies. Due to improvements in expression technology and optimization of fermentation, culture supernatants with high antibody content must be processed. Recently protein A affinity media with improved adsorption characteristics have been developed. The agarose media MabSelect Xtra and MabSelect SuRe are recent developments of the existing protein A affinity medium MabSelect. MabSelect Xtra is designed to exhibit a higher binding capacity for IgG, and MabSelect SuRe is functionalized with an alkaline stabilized protein A. ProSep-vA Ultra is a porous glass medium with a pore size of 70 nm, also developed to improve the binding capacity. Adsorption was measured in a finite and infinite bath. Mass transfer in these systems could be well described by a model including film and pore diffusion. Mass transfer parameters were used to accurately predict IgG breakthrough in packed bed mode. The dynamic binding capacity of all three media did not change when residence time was at least 4 min. All three media are suited for capture of feed stocks with high antibody content.

Chromatography, Affinity↗

PubMed2005

Hydrophobic interaction chromatography of proteins. III. Unfolding of proteins upon adsorption.

Hydrophobic interaction chromatography (HIC) exploits the hydrophobic properties of protein surfaces for separation and purification by performing interactions with chromatographic sorbents of hydrophobic nature. In contrast to reversed-phase chromatography, this methodology is less detrimental to the protein and is therefore more commonly used in industrial scale as well as in bench scale when the conformational integrity of the protein is important. Hydrophobic interactions are promoted by salt and thus proteins are retained in presence of a cosmotropic salt. When proteins are injected on HIC columns with increasing salt concentrations under isocratic conditions only, a fraction of the applied amount is eluted. The higher the salt concentration, the lower is the amount of eluted protein. The rest can be desorbed with a buffer of low salt concentration or water. It has been proposed that the stronger retained protein fraction has partially changed the conformation upon adsorption. This has been also corroborated by physicochemical measurements. The retention data of 5 different model proteins and 10 different stationary phases were evaluated. Partial unfolding of proteins upon adsorption on surfaces of HIC media were assumed and a model describing the adsorption of native and partial unfolded fraction was developed. Furthermore, we hypothesize that the surface acts as catalyst for partial unfolding, since the fraction of partial unfolded protein is increasing with length of the alkyl chain.

Adsorption↗

PubMed2005

In situ determination of adsorption kinetics of proteins in a finite bath.

A method for fast in situ measurement of adsorption kinetics based on a finite bath was developed. We modified the conventional finite bath by replacing the external loop by a dip probe which enables in situ measurement of the concentration change in the contactor. Deposition of adsorbent particles on the reflection surface of the dip probe compromised measurements. Different membranes, a polyamide, a polypropylene and a nylon membrane were tested to protect the internal reflection surface of the dip probe from fouling with adsorbent particles. The nylon membrane provided efficient protection and high mass transfer evaluated by response time experiments. Unspecific adsorption of the model protein on the membrane could also be excluded. To corroborate the measurements of the dip probe the results were compared to a conventional finite bath and to a shallow-bed. The uptake curves for human polyclonal IgG at different concentrationes (0.1-3 g/l) on rProtein A Sepharose FF and MabSelect were used as model system. The effective diffusion coefficients were determined using a pore diffusion model. These values were in good agreement for all methods.

Adsorption↗

PubMed2004

Performance and characterization of a nanophased porous hydroxyapatite for protein chromatography.

Nanophased porous hydroxyapatite beads with particle diameters of 25 microm and 30 microm intended for use in protein and biomolecule separation are characterized with respect to chromatographic characteristics. These particles were produced from a hydroxyapatite gel by a controlled spray process yielding microspheres containing hydroxyapatite nanocrystals. By calcification of the microspheres, nanophased porous hydroxyapatite beads were obtained. As a reference material, ceramic hydroxyapatite Types I and II with a particle diameter of 40 microm was chosen. SEM pictures show that the surface of the nanophased hydroxyapatite is very rough compared to ceramic hydroxyapatite Types I and Type II. The calcium-to-phosphorous ratio of this nanophased hydroxyapatite is 1.6, which is slightly below the theoretical ratio of 1.67 of pure hydroxyapatite. The porosity is greater than 60%. An IgG binding capacity of 60.7 mg/ml for Bio-Rad Type I and 36.0 mg/ml for Type II, 42.0 mg/ml for the nanophased material with 25 microm and 19.7 mg/ml for the nanophased material with 30 microm were observed. The nanophased material with 30 microm had the lowest mass transfer resistancy as indicated by the dependency of the dynamic binding capacity on velocity. It is assumed that the mass transport properties are characterized by a low particle diffusion resistancy or by slight intraparticle convection. The material also showed high selectivity for IgG. When culture supernatant with 5% FCS containing 3 mg/ml was loaded, pure IgG could be eluted by linear gradient with increasing sodium phosphate concentration. This nanophased material comprises a novel stationary phase for IgG separation.

Adsorption↗

PubMed2004

Mass transfer characteristics of plasmids in monoliths.

The hydrodynamic properties and pore-structure of monoliths based on functionalized poly(glycidyl methacrylate-ethylene dimethacrylate) were characterised by pulse response experiments using different probes representing a wide range of molecular mass. On a small scale, band spreading was found to be caused to the extent of more than 90% by extra-column effects. These monoliths have large channel diameters, providing a suitable chromatography adsorbent for processing of large molecules. Dynamic and static binding capacity for plasmid DNA was investigated. For our model plasmid, consisting of 4.9 kbp, a capacity of 7 mg/mL was observed in comparison to 0.3 mg/mL for a conventional medium designed for protein separation. When plasmids were loaded on the monolith a gradual increase in pressure drop was observed. The channels filled up and the cross-sectional area available for liquid flow decreased. Therefore, a higher pressure drop was observed during elution. This is caused by (i) shrinking of the channels as effect of the high salt concentration, (ii) high viscosity of the mobile phase due to high concentration of plasmids, and (iii) an increase of the hydrodynamic radius of the plasmid with salt concentration from 45 nm at 150 mM to 70 nm at 2 M NaCl, as measured by dynamic light scattering. These types of monoliths are considered to be the preferred adsorbents for plasmid separation.

Adsorption↗

PubMed2004

Monoliths for fast bioseparation and bioconversion and their applications in biotechnology.

Monoliths have consolidated their position in bioseparation. More than 200 different applications have been reported in the past two decades and their advantages compared to conventional chromatography demonstrated. These include the high mass transfer efficiency due to the convective flow enabled by the macroporous character of the matrix. Recently plasmid DNA and viruses were separated with high efficiency and cryogels and monolithic superporous agarose were developed for capture of proteins from crude homogenates and separation of microorganisms or lymphocytes. Currently four companies manufacture monoliths mainly for analytical applications although monoliths with a volume of 0.8 liter are commercially available and 8 L are available as prototypes. A book entitled "Monolithic materials: preparation, properties and applications" was published in 2003 and became standard reference of the status of this area. This review focuses on the progress in monoliths that goes beyond the scope of this reference book. Less progress has been made in the field of bioconversions in spite of the fact that monolithic supports exhibit better performance than beads in enzymatic processing of macromolecules. It appears that the scientific community has not yet realized that supports for these applications are readily available. In addition, monoliths will further substantially advance bioseparations of both small and large molecules in the future.

Animals↗

PubMed2004

Engineering protein A affinity chromatography.

Staphylococcal protein A can selectively interact with immunoglobulins. This protein is widely used as a ligand for affinity chromatography to purify therapeutic antibodies on an industrial scale. This type of affinity chromatography constitutes a generic step in processing antibodies. Questions of scale-up, design of chromatographic conditions, clearance of adventitious agents and operational modes, such as continuous operation or purification of antibodies in expanded-bed mode, will be addressed in this review.

Antibodies, Monoclonal↗

PubMed2003

Hydrophobic interaction chromatography of proteins. II. Binding capacity, recovery and mass transfer properties.

Hydrophobic interaction chromatography media suited for large scale separations were compared regarding dynamic binding capacity, recovery and mass transfer properties. In all cases, pore diffusion was the rate limiting step. Reduced heights equivalent to a theoretical plate for bovine serum albumin derived from breakthrough curves at reduced velocities between 60 and 1500 ranged from 10 to 700. Pore diffusion coefficients were derived from pulse response experiments for the model proteins alpha-lactalbumin, lysozyme, beta-lactoglobulin, bovine serum albumin and immunoglobulin G. Diffusivity of lysozyme did not follow the trend of decreasing diffusivity with increasing molecular mass, as observed for the rest of the proteins. In general, mass transfer coefficients were smaller compared to ion-exchange chromatography. Dynamic binding capacities for the model protein bovine serum albumin varied within a broad range. However, sorbents based on polymethacrylate showed a lower dynamic capacity than media based on Sepharose. Some sorbents could be clustered regarding binding capacity affected by salt. These sorbents exhibited a disproportional increase of binding capacity with increasing ammonium sulfate concentration. Recovery of proteins above 75% could be observed for all sorbents. Several sorbents showed a recovery close to 100%.

Chromatography, Liquid↗

PubMed2003

Comparison of protein A affinity sorbents.

Protein A is a popular generic ligand for purification of monoclonal and recombinant antibodies. The performance of 15 commercially available protein A media was studied. Equilibrium and dynamic binding capacity for human IgG was determined and the capture of IgG from a crude feed-stock was investigated. For initial screening the dynamic binding capacity was determined at small scale. Media with good performance were further tested with increased column height. Comparing the data from the two different column heights it could be shown that the dynamic capacity strongly depends on the residence time. Agarose based media exhibited higher binding capacity at residence times longer than 3 min whereas polymeric media or media based on porous glass showed a lesser dependence on the flow velocity and the residence time. A quantitative description of this behavior was derived by determination of the adsorption isotherms and fitting the breakthrough profiles with the Thomas solution. Agarose based media exhibited higher maximum equilibrium binding capacities and the dissociation constants derived from adsorption isotherms were smaller. The other media exhibited higher apparent rate constants, indicating a faster mass transfer. This can be explained by the smaller particle diameter of these media and it can be assumed that constant pattern conditions are thereby obtained more quickly. Selectivity was tested by performing antibody purification under standardized conditions. Polyclonal human IgG in cell culture supernatant containing 2.5% fetal calf serum was used as a representative feed-stock. Under the applied conditions several sorbents showed very tight binding of IgG and in some cases most of the sample remained on the sorbent. The study can be useful as a guide for optimization of large-scale purification processes.

Adsorption↗

PubMed2003

Directed immobilization of peptide ligands to accessible pore sites by conjugation with a placeholder molecule.

When small ligands are immobilized onto a porous chromatography medium, only a limited number of binding sites contributes to the interaction with the target molecule. The main part of the ligand molecules is distributed on sites that are not accessible for the target protein due to steric hindrance. To direct the ligand into a well-accessible position, the ligand was conjugated to a large molecule that acted as a placeholder during the immobilization step. Then the placeholder molecule was cleaved off and washed out. Two linear peptides with affinity for lysozyme and human blood coagulation factor VIII, respectively, were studied as model systems. The protected peptide ligand was covalently linked to a 20-kDa poly(ethylene glycol) molecule containing an acid-labile linker. After selective deprotection of the peptide and purification, immobilization of this conjugate on a preactivated chromatography matrix was performed alternatively through the free N-terminus, the epsilon-amino group of lysine, or the sulfohydryl group of cysteine. After the immobilization reaction, the spacer molecule and remaining protecting groups were cleaved off and the gels were tested by affinity chromatography. This novel immobilization technique substantially increased the binding capacity and the ligand utilization for the target protein, and site-specific immobilization could be demonstrated.

Chromatography, Affinity↗

PubMed2002

Hydrophobic interaction chromatography of proteins. I. Comparison of selectivity.

Currently, the selection of a hydrophobic interaction chromatography (HIC) sorbent for protein separation purposes is entirely based on empirical means. An attempt was made to characterize different HIC sorbents from various manufacturers. The selectivity was determined by isocratic pulse experiments of a set of reference proteins and an algorithm was developed to classify the sorbents according to their selectivity and hydrophobicity. The obtained semi-quantitative parameters take into account the dependence of salt on adsorption. The sorbent characteristics evaluated with the model proteins were compared to the separation of a real feedstock. A good agreement was achieved between the developed evaluation procedure and the separation behaviour of the real feed stock.

Chromatography, Liquid↗