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Biomedical subjects

R Freitag

Publications and source records attributed to R Freitag.

At least 19 recordsLinked to original sources
PubMed2002

On-line microdialysis of proteins with high-salt buffers for direct coupling of electrospray ionization mass spectrometry and liquid chromatography.

Mass spectrometry (MS) is one of the most powerful instrumental techniques for protein analysis. The electrospray ionization (ESI) approach is known to be very gentle and at the same time compatible with liquid separation techniques such as HPLC and CE. However, ESI is known to be susceptible to salts and impurities, which often cause a dramatic decrease in sensitivity due to the suppression of the ionization of the product of interest. For this reason, LC-ESI-MS coupling has so far been largely limited to reversed-phase chromatography with its hydro-organic mobile phases. Other chromatographic techniques are typically "linked" to ESI-MS by time consuming, off-line desalting steps. On-line microdialysis has been proposed as a solution to this dilemma. In this paper, we introduce an improved microdialysis system, which enlarges the number of putative applications, thus allowing chromatographic separations of biological compounds to be directly coupled to MS detection with little to no loss in time or chromatographic resolution. Examples include separations by affinity, ion-exchange and size-exclusion chromatography, all of which were connected successfully to the ESI-MS detector via the on-line microdialyzer. We propose that, using this system, any kind of chromatography technique can be coupled to ESI-MS, thus enabling for example application in quality control or process monitoring of many bioproduction and downstream processes.

Animals↗
PubMed2002

Continuous isolation of plasmid DNA by annular chromatography.

Continuous chromatographic separations, especially of multicomponent mixtures, constitute interesting options for biotechnological downstream processing. Taking the separation of plasmid DNA from clearified lysates on hydroxyapatite as a pertinent example, we discuss the potential of continuous annular chromatography (CAC) in comparison with conventional (preparative) batch chromatography. In CAC the column is realized in the form of a thin (5 mm, height 210 mm) slowly rotating annulus. The performance of such a CAC column is compared to that of an ("analytical") batch column of similar thickness (diameter) and length (4 x 250 mm) and that of a ("preparative") batch column of similar cross-sectional surface area and height (50 x 210 mm). The quality of the obtained plasmid as defined by the appearance of the corresponding agarose gels (native and linearized plasmid), the 260/280 ratio and the biological activity (transient transfection of HEK 293 cells) was found to be identical in all three cases. The yields are also shown to be equivalent. The loading factor is found to be the most decisive parameter for the transfer of a given separation method between the continuous and the batch columns. Under nonoptimized conditions, plate numbers tended to be lower in the continuous compared to the batch columns. This is shown to be largely due to an artifact created by the CAC design (collection of averaged fractions at the outlets) and can be overcome by optimizing the rotation speed. Surprisingly the large batch column consistently gave better plate numbers than either the small batch or the CAC column. Compared to the preparative batch column, wall effects are more pronounced in the CAC (respectively the small diameter batch column), which may translate into better bed stability but conceivably also contributes to an increase in plate height, due to the reduction in bed density usually observed in the proximity of the wall. The CAC is shown to be a powerful approach to continuous chromatography, which allows a direct and straightforward upscale of chromatographic bioseparation methods.

Cell Line↗
PubMed2002

Mass influences in the performance of oligomeric poly(diallyldimethylammonium chloride) as displacer for cation-exchange displacement chromatography of proteins.

A novel type of linear polyelectrolyte, namely poly-DADMAC [poly(diallyldimethylammonium chloride)], was prepared and studied as a displacer for cation-exchange displacement chromatography of proteins. In contrast to the commercially available polymers of that chemistry, the novel type of poly-DADMAC introduced here is characterized by a homogeneous linear structure, a narrow distribution of the (adjustable) molar mass as well as by a defined and homogeneous affinity for the stationary phase. Five poly-DADMACs of different size (17900 to 88000 g/mol) were prepared and compared with regard to their stationary phase affinity and protein separation potential, taking a mixture of basic proteins, namely lysozyme, cytochrome C, and ribonuclease A (from bovine pancreas), as an example. The steric mass action model was employed to aid method development. Under the chosen conditions (low ionic strength of the mobile phase guaranteeing strong binding of both the proteins and the displacer) the poly-DADMAC with the lowest molar masses proved to be the most efficient displacers for the basic proteins with a stationary phase affinity constant of 5.3 x 10(16) and a steric factor of 224. Using this substance as displacer, a sample mixture containing up to three proteins was separated and the proteins recovered at high yields (80-97%) and in high purity and concentration.

Cation Exchange Resins↗
PubMed2001

Isolation of a recombinant antibody from cell culture supernatant: continuous annular versus batch and expanded-bed chromatography.

Annular chromatography represents a crossflow approach to chromatographic separations, that allows the continuous separation of multicomponent mixtures. The potential of the method for continuous bioseparation has been discussed for some time, however, we demonstrate for the first time the processing of a complex feed (cell culture supernatant) taken from an actual (bio)process. Moreover, while previously published applications of annular chromatography concentrated on noninteractive (gel filtration) or nonspecific (ion exchange) chromatography, we show the possibility of continuous annular affinity chromatography. In particular, a commercially available preparative continuous annular chromatography (P-CAC) system was used to purify a recombinant antibody (human IgG(1)-kappa) from CHO cell culture supernatants by (pseudo)affinity chromatography on hydroxyapatite (HA) and rProtein A. Methods developed using small (2 mL) batch columns could be directly transferred to the P-CAC, where they yielded similar results in terms of final product quality. Yields were between 87% and 92% in the case of HA and between 77% and 82% in the case of rProtein A chromatography. DNA removal was nearly quantitative in all cases. Concomitantly, the antibody fraction of the total protein content was raised by one order of magnitude in HA and by a factor of 50 by rProtein A chromatography. In addition, a novel HA material (particle diameter -120 microm) was investigated, which was compatible with expanded-bed applications. However, the final purity of the antibody thus obtained and also the yields (<70%) were less than satisfactory.

Animals↗
PubMed2001

Acrylamide-based monoliths as robust stationary phases for capillary electrochromatography.

A method is described for the synthesis of rigid, macroporous polymers (monoliths) to be used as stationary phases in capillary electrochromatography (CEC). The procedure reproducibly results in columns with good mechanical and chemical stability. Once the procedure was optimized, it yielded the desired CEC columns in nearly 100% of the cases. The batch-to-batch standard deviation of the migration of the electroosmotic flow (EOF) marker for nine randomly chosen columns was 5%. The polymerization is carried out inside the capillary, an aqueous phase is used as solvent. Monomers based on acrylamides with varying hydrophilicity were used to introduce the interactive moieties together with piperazine diacrylamide as cross-linker and vinylsulfonic acid as provider of the charged, EOF-producing moieties. The pore size of the monoliths was adjusted by adding varying amounts of ammonium sulfate to the reaction mixture. In this manner, the average pore size of a given monolith could be reproducibly adjusted to values ranging from 50 nm to 1.3 microm. The procedure was optimized for four particular types of monoliths, which differed in hydrophobicity. The latter was adjusted by introducing suitable co-monomers, such as alkyl chain-bearing molecules, into the monolithic structure. Attempts to systematically investigate the chromatographic behavior of the monolithic stationary phases were made, using a model mixture of aromatic compounds as sample. The standard deviations for the run-to-run reproducibility of the retention times for unretained and retained analytes were <1.5%. Flat Van Deemter curves were measured even at elevated flow-rates (2 mm/s). Plate heights between 10 and 15 microm were measured in this range. The retention order was taken as the principal indication for the chromatographic mode. The separation was found to be governed neither by pure reversed-phase nor by pure normal-phase chromatography, even on monoliths, where large amounts of C6 ligands had been introduced.

Acrylamides↗
PubMed2001

Different subsets of enteric bacteria induce and perpetuate experimental colitis in rats and mice.

Resident bacteria are incriminated in the pathogenesis of experimental colitis and inflammatory bowel diseases. We investigated the relative roles of various enteric bacteria populations in the induction and perpetuation of experimental colitis. HLA-B27 transgenic rats received antibiotics (ciprofloxacin, metronidazole, or vancomycin-imipenem) in drinking water or water alone in either prevention or treatment protocols. Mice were treated similarly with metronidazole or vancomycin-imipenem before or after receiving 5% dextran sodium sulfate (DSS). Germfree transgenic rats were colonized with specific-pathogen-free enteric bacteria grown overnight either in anaerobic or aerobic atmospheres. Nontransgenic rats colonized with anaerobic bacteria served as negative controls. Although preventive metronidazole significantly attenuated colitis in transgenic rats and DSS-treated mice, it had no therapeutic benefit once colitis was established. Ciprofloxacin also partially prevented but did not treat colitis in B27 transgenic rats. In both animal models vancomycin-imipenem most effectively prevented and treated colitis. Germfree transgenic rats reconstituted with enteric bacteria grown under anaerobic conditions had more aggressive colitis than those associated with aerobic bacteria. These results suggest that a subset of resident luminal bacteria induces colitis, but that a complex interaction of commensal aerobic and anaerobic bacteria provides the constant antigenic drive for chronic immune-mediated colonic inflammation.

Animals↗
PubMed2000

Preparation of stationary phases for open-tubular capillary electrochromatography using the sol-gel method.

Capillary electrochromatography requires the deposition of a stationary phase inside the capillary. In this paper the sol-gel method is proposed for this purpose. The gels were prepared externally and injected into a fused-silica capillary, where anchorage to the capillary wall was possible through condensation reactions between the silanol groups of the capillary wall and the residual silanol groups the gel. Contrary to a commonly used practice, alkaline pretreatment of the inner capillary wall prior to the introduction and anchoring of the gel was found to be only marginally effective in improving the mechanical stability of the column. The influence of various parameters, such as the pH, the water content, the presence of alcohol (ethanol) on the formation of tetraethoxysilane (TEOS)-n-octyltriethoxysilane (C8-TEOS) hybrid gels of varied composition is discussed. The pH and the amount of water present were found to be the determining factors in the preparation of a stable gel with the desired mechanical and chromatographic properties. By carrying out the gel formation at 80 degrees C, capillary columns could be produced in 2.5 h. While an acidic pH was required during (external) gel formation, subsequent treatment of the gel inside the capillary with an alkaline solution ('aging') was found to improve separation and stationary phase capacity significantly. The capillary columns were subsequently used to separate a mixture of polycyclic aromatic hydrocarbons in less than 3 min.

Electrophoresis, Capillary↗
PubMed2000

Comparison of particulate and continuous-bed columns for protein displacement chromatography.

A conventional anion exchange column packed with porous particles (BioScale Q2), and a novel continuous-bed column (UNO Q1) were compared for displacement separation of dairy whey proteins with polyacrylic acid as displacer. The steric mass action model was investigated as a means to aid and accelerate this development. Characteristic charges and steric factors were measured for the proteins and the displacer according to the model, and used together with the affinity constant derived from the adsorption isotherms for simulations, as well as for the construction of the affinity and operating regime plots. If possible, the latter two were used to select conditions for the actual experiments. In the case of the particle-based column, experimental results and simulations did not agree. In addition, the operating regime plot could not be constructed. The affinity plot did predict the order in the displacement train correctly, but gave misleading information concerning the possible effect of a change in displacer concentration. This is taken to be a result of the porous nature of the particles, which handicaps, to some extent, the interaction of the proteins and the displacer molecules with the adsorptive surface. Results were considerably better in case of the continuous-bed column, where there is no intraparticulate surface.

Acrylic Resins↗
PubMed2000

Comparison of antibody binding to immobilized group specific affinity ligands in high performance monolith affinity chromatography.

A novel biochromatographic principle is introduced taking the quantitative analysis of affinity interactions between antibodies and immobilized group specific ligands (protein A, G, and L) as example. The name high performance monolith affinity chromatography (HPMAC) is proposed for this technique. HPMAC uses rigid, macroporous monoliths, so-called convective interaction media (CIM)-disks, as stationary phase. An optimized procedure is described for the covalent immobilization of the group specific affinity ligands to such disks. The binding of polyclonal bovine IgG and a recombinant human antibody (type IgGl-kappa) to all affinity disks is discussed. An essential feature of HPMAC is its compatibility to unusually high mobile phase flow rates ( > 4 ml/min). Chromatographic experiments are thus completed within seconds without significant loss in binding capacity and retentive power. This makes HPMAC a promising tool for applications in fast process monitoring or screening. As an example for the former, the direct quantitative isolation of recombinant antibodies from serum-free culture supernatant is demonstrated.

Animals↗
PubMed2000

Comparison of different types of ceramic hydroxyapatite for the chromatographic separation of plasmid DNA and a recombinant anti-rhesus D antibody.

The chromatographic properties of three types of ceramic hydroxyapatite (HAP) are compared. All three materials were prepared by sintering the original precipitate, albeit at different temperatures (400, 700 and 1000 degrees C for type I, II and III HAP, respectively). The three materials differed in pore size and pressure limits (both lowest for type I and highest for type III). Type I and II HAP had an average particle diameter of 20 microm. The particle size of the type III material was 40 microm. HAP-beads were slurry-packed into 4 x 25-mm stainless steel columns and investigated for the chromatographic isolation of plasmid DNA from clarified E. coli lysates and of a recombinant human antibody from CHO cell culture supernatants respectively. The chromatographic performance of the three types of HAP showed significant differences, which were correlated to the binding capacities of the materials for (linearized) plasmids of different size (4.7, 10.3 and 11.4 kb) and proteins of different isoelectric point (lysozyme, pI = 10.5; anti RhD antibody, pI = 8.3; beta-lactoglobulin, pI = 4.9). The accessibility of the adsorptive surface (pore size) but also the types of binding sites on the HAP-surface (P/C-site ratio) are proposed as determining factors for the chromatographic behavior.

Animals↗
PubMed1999

Polycations as displacer in high-performance bioseparation.

Displacement chromatography is an interesting but up to now rarely used type of preparative biochromatography. The lack of well-engineered and accessible displacer contributes to this phenomenon. In this paper a novel type of displacer is introduced for cation-exchange displacement chromatography, which will soon become commercially available. The molecule is a well-defined PolyDADMAC [poly(diallyldimethylammonium chloride)] with a molar mass of less than 35000 g/mol, an exclusively linear structure and a molar mass polydispersity of less than 1.5. A method for synthesizing such a polymer at high yields is described. The PolyDADMAC is shown to be an efficient displacer of basic proteins from strong cation-exchange columns.

Chromatography, Ion Exchange↗
PubMed1999

High performance flow injection analysis of recombinant protein G.

Chromatographic discs were investigated for their potential to substitute for the hitherto used cartridges in heterogeneous flow injection analysis. Originally designed for fast high performance liquid chromatography (HPLC) of biopolymers, the discs combine reliability with speed and resolution. This together with their price and their long-standing time made them attractive for use in flow injection analysis. The base material of the discs is a glycidyl methacrylate-co-ethylene dimethacrylate (GMA-EDMA) co-polymer. The epoxy groups inherent to this base structure can be used for immobilization purposes. In this first demonstration, antibodies were immobilized and the resulting affinity discs used for the fast analysis (< 5 min) of protein G from cell lysate of recombinant Escherichia coli. A linear calibration curve over several orders of magnitude as well as excellent reproducibility and correlation with data produced by conventional protein assay were obtained.

Chromatography↗
PubMed1999

Utilization of enzyme-substrate interactions in analytical chemistry.

Enzymes are capable of a highly specific interaction with a variety of substances including their respective substrates. This review summarizes how such interactions may be used in analytical (bio-)chemistry, e.g., for the elucidation of the binding mechanism, the determination of the binding strength, the carting of the binding site, or the screening of possible substrate/inhibitor molecules. Possible assay formats such as analytical affinity chromatography, affinity capillary electrophoresis (ACE), conventional affinity gel electrophoresis (AEP), and related techniques are discussed together with examples of recent applications. In addition a brief section on enzyme-substrate reactions as tools in analytical chemistry is included, since these are perhaps even more important to analytical (bio-)chemistry. The development and application of bioanalytical systems and especially biosensors in various fields including medicine, biotechnology, agriculture, defense and foodstuffs are considered.

Electrophoresis↗
PubMed1999

Thermoreactive displacers for anion exchange and hydroxyapatite displacement chromatography

A set of copolymers was prepared via radical copolymerization of N, N-dimethylacrylamide and 7-hydroxy-3-methyl-4-vinylindanone. While poly(N,N-dimethylacrylamide) is water-soluble at all temperatures, the copolymers showed a lower critical solution temperature (LCST), which depended on the composition. Independent of this LCST all copolymers can be used in the dissolved state as protein displacers in combination with anion exchange stationary phases. The indanone group of the polymers is known the chelate calcium ions. It was therefore also possible to use the polymers to displace acidic (i.e., C-site interacting) proteins from hydroxyapatite. The major advantage of the described displacers over existing agents stems from the fact that they can be almost quantitatively recovered by thermoprecipitation from the carrier but also from any given protein zone. Lowering of the temperature renders the polymers again fully soluble and ready for use. No unspecific protein coprecipitation was observed under these conditions within our analytical means.

Journal Article↗
PubMed1998

High-performance membrane chromatography of supercoiled plasmid DNA.

Membrane adsorbers are well established in protein chromatography. The present paper investigated for the first time the behavior of polynucleotides on these stationary phases, taking a 7.2-kb predominantly supercoiled plasmid as example. Gradient and isocratic elution was studied. In contrast to protein high-performance membrane chromatography (HPMC), isocratic elution is possible in DNA chromatography. In the case of gradient elution, much higher salt concentrations can be used in the starting buffer. Under optimized conditions, both approaches led to a splitting of the single plasmid peak into three maximums, which corresponded to the three-albeit isolated-bands in the agarose gel. Presumably the three fractions were supercoiled, nicked, and open circular plasmid DNA. Linearization of the plasmid lowered the adsorption energy, and the linearized plasmid eluted earlier than the nonlinearized one. The HPMC experiments were compared to similar ones performed using a conventional packed-bed anion-exchange column (BioScale Q2, 7 x 52 mm, 10-micron porous particles) and a novel monolithic-type anion-exchange column (UNO Q1, 7 x 35 mm). The results and characteristic differences observed in these experiments were interpreted in the light of the newly developed theory of HPMC.

Chromatography↗
PubMed1998

Fast isolation of protein receptors from streptococci G by means of macroporous affinity discs.

A fast affinity method for the semi-preparative isolation of recombinant Protein G from E. coli cell lysate is proposed. Rigid, macroporous affinity discs based on a glycidyl methacrylate-co-ethylene dimethacrylate polymer were used as chromatographic supports. The specific ligands (here human immunoglobulin G, hIgG) were immobilized by the one-step reaction between native epoxy groups of the polymer surface and epsilon-amino groups of the IgG molecules. No intermediate spacer was necessary to reach full biological activity of the ligand. The globular affinity ligands are located directly on the pore wall surface and are thereby freely accessible to target molecules (here Protein G) migrating with the mobile phase through the pores. It is shown that the conditions chosen for the hIgG immobilization do not involve an active site of the protein and thus do not bias the formation of the affinity complex. Chromatographically determined constants of dissociation of hIgG-Protein G affinity complexes confirm the high selectivity of this separation method. Two different aspects of the affinity separation are discussed, which differ mostly in terms of scale. In disc chromatography, high volumetric flow velocities are possible because of the small backpressure. Since in addition the mass transfer is more efficient, it becomes possible to achieve very short analysis times. The discs proposed can be used in a single-step enrichment of Protein G from lysates of non-pathogenic E. coli. Gel electrophoresis data are used to demonstrate the high degree of purity achieved for the final product.

Bacterial Proteins↗
PubMed1998

Development of a downstream process for the isolation and separation of monoclonal immunoglobulin A monomers, dimers and polymers from cell culture supernatant.

The isolation and separation of the molecular variants of monoclonal IgA from cell culture supernatants is possible using several filtration and ion-exchange chromatography steps, followed by size-exclusion chromatography for the actual separation of the molecular variants. The latter step is especially time consuming and laborious. This report presents possible improvements of the procedure. Use of the displacement rather than the elution mode may render the ion-exchange step more productive (higher product concentrations and space-time yield). For the final separation of the molecular variants, hydroxyapatite (HA) elution chromatography can serve as an alternative to size-exclusion chromatography. By using an optimized, complex phosphate gradient, the IgA dimers can be separated quantitatively from the monomers and higher oligomers. It may in individual cases be necessary to use a size-exclusion polishing step to reach the required final degree of purity, however, the amount of material to be processed is reduced to such an extend by the HA-step, that the overall process is still more productive. Buffer pH and flow-rate as well as the stationary phase material used were additional factors considered during the optimization of the HA elution chromatography. HA-displacement chromatography resulted only in a concentration of the overall IgA fraction, but not in a separation of the molecular forms.

Animals↗