Biomedical subjects

Ruth Freitag

Publications and source records attributed to Ruth Freitag.

8 recordsLinked to original sources

PubMed2003

Investigation of particle-based and monolithic columns for cation exchange protein displacement chromatography using poly(diallyl-dimethylammonium chloride) as displacer.

The overall topic of the investigation was the separation of basic proteins by cation exchange displacement chromatography. For this purpose two principal column morphologies were compared for the separation of ribonuclease A and alpha-chymotrypsinogen, two proteins found in the bovine pancreas. These were a column packed with porous particles (Macro-Prep S, 10 microm, 1000 A) and a monolithic column (UNO S1). Both columns are strong cation exchangers, carrying -SO3(-)-groups linked to a hydrophilic polymer support. Poly(diallyl-dimethylammonium chloride) (PDADMAC), a linear cationic polyelectrolyte composed of 100-200 quaternary pyrrolidinium rings, was used as displacer. The steric mass action (SMA) model and, in particular, the operating regime and dynamic affinity plots were used to aid method development. To date the SMA model has been applied primarily to simulate non-linear displacement chromatography of proteins using low molar mass displacers. Here, the model is applied to polyelectrolytes with a molar mass below 20000 g mol(-1), which corresponds to a degree of polymerization below 125 and an average contour length of less than 60 nm. The columns were characterized in terms of the adsorption isotherms (affinity, capacity) of the investigated proteins and the displacer.

Cation Exchange Resins↗

PubMed2003

Investigation of mixed-mode monolithic stationary phases for the analysis of charged amino acids and peptides by capillary electrochromatography.

The potential of N,N-dimethylacrylamide-piperazine diacrylamide-based monolithic stationary phases bearing sulfonic acid groups for electroosmotic flow generation is investigated for the separation of positively charged amino acids and peptides. The capillary columns were used under electrochromatographic but also under purely chromatographic (nano-HPLC) conditions and the separations interpreted as the result of possible chromatographic and electrophoretic contributions. The stationary phases were found to be mechanically stable up to pressures of 190 bar and chemically stable towards a wide variety of organic and hydro-organic mobile phases. In order to investigate the retention mechanism, the salt concentration and the organic solvent content of the (hydro-)organic mobile phase were varied in a systematic manner, taking three aromatic amino acids (phenylalanine, tryptophan, histidine) as model analytes. The respective contributions of electrostatic and hydrophobic and/or hydrophilic interactions were further investigated by varying the charge density and the hydrophobicity of the standard stationary phase. The former was done by varying the amount of charged monomer (vinylsulfonic acid) added during synthesis, the latter by (partially) replacing the interactive monomer (N,N-dimethylacrylamide) by other more hydrophobic monomers. A mixed mode retention mechanism based primarily on electrostatic interactions modified in addition by "hydrophilic" ones seems most suited to interpret the behavior of the amino acids, which stands in contradistinction to the previously investigated case of the behavior of neutral analytes on similar stationary phases. Finally the separation of small peptides was investigated. While the separation of Gly-Phe and Gly-Val was not possible, the separation of Phe-Gly-Phe-Gly and Gly-Phe but also of the closely related Gly-His and Gly-Gly-His could be achieved.

Amino Acids↗

PubMed2003

Protein purification by affinity precipitation.

Developing the most efficient strategy for the purification of a (recombinant) protein especially at large scale remains a challenge. A typical problem of the downstream process of mammalian cell products is, for instance, the early capture of the highly diluted product from the complex process stream. Affinity precipitation has been suggested in this context. The technique is known for over 20 years, but has recently received more attention due to the development of new materials for its implementation, but also because it seems ideally suited to specific product capture at large scale. The present review gives a comprehensive overview over this technique. Besides an introduction to the basic principle and a brief summary of the historical development, the main focus is on the current state-of-art of the technique, the available materials, important recent applications, as well as process design strategies and operating procedures. Special consideration is given to affinity precipitation for product recovery at large scale.

Chromatography, Affinity↗

PubMed2003

Continuous annular chromatography.

The principle of continuous annular chromatography (CAC) has been known for several decades. CAC is a continuous chromatographic mode, which lends itself to the separation of multi-component mixtures as well as of bi-component ones. In CAC, the mobile and stationary phases move in a crosscurrent fashion, which allows transformation of the typical one-dimensional batch column separation into a continuous two-dimensional one. With the exception of linear gradient elution, all chromatographic modes have at present been applied in CAC. This review focuses on the capacity of CAC for preparative bioseparation. The historical developments and the predecessors of modern CAC are briefly summarized. The state-of-the-art in the theoretical prediction and simulation of CAC separations is discussed, followed by an overview of current CAC instrumentation and example applications, especially for the isolation of proteins and other bio(macro)molecules. In this context, issues of scale up as well as method development and transfer from batch to continuous CAC columns are discussed using recent bioseparation efforts as pertinent examples.

Animals↗

PubMed2003

DNA purification by triple-helix affinity precipitation.

Recent advances in DNA-based medicine (gene therapy, genetic vaccination) have intensified the necessity for pharmaceutical-grade plasmid DNA purification at comparatively large scales. In this contribution triple-helix affinity precipitation is introduced for this purpose. A short, single-stranded oligonucleotide sequence (namely (CTT)(7)), which is capable of recognizing a complementary sequence in the double-stranded target (plasmid) DNA, is linked to a thermoresponsive N-isopropylacrylamide oligomer to form a so-called affinity macroligand (AML). At 4 degrees C, i.e., below its critical solution temperature, the AML binds specifically to the target molecule in solution; by raising the temperature to 40 degrees C, i.e., beyond the critical solution temperature of the AML, the complex can be precipitated quantitatively. After redissolution of the complex at lower temperature, the target DNA can be released by a pH shift to slightly alkaline conditions (pH 9.0). Yields of highly pure (plasmid) DNA were routinely between 70% and 90%. Non-specific co- precipitation of either the target molecule by the non-activated AML precursor or of contaminants by the AML were below 7% and presumably due to physical entrapment of these molecules in the wet precipitate. Ligand efficiencies were at least 1 order of magnitude higher than in triple-helix affinity chromatography.

Affinity Labels↗

PubMed2003

Investigation of conditions allowing the synthesis of acrylamide-based monolithic microcolumns for capillary electrochromatography and of factors determining the retention of aromatic compounds on these stationary phases.

The influence of the cross-linker (concentration), the porogen (lyotrophic salt) and the solvent type as well as the type and concentration of up to three "functional", i.e., interactive monomers on the morphology and the chromatographic properties of acrylamide-based hydrophilic monoliths are investigated. High total monomer concentrations favored polymers with a rigid rather than gel-like structure. High cross-linker concentrations also favor the formation of a nodular structure. The addition of a lyotrophic salt favors the formation of small nodules especially at higher monomer concentration; the pore size of the polymer can also be modulated through the salt concentration. Suitable monoliths were further investigated as potential stationary phases for capillary electrochromatography (CEC). Depending on the type and concentration of the monomers, plate numbers between 50,000 and 100,000 were routinely obtained. The standard deviation of the run-to-run reproducibility was below 2% and that of the batch-to-batch reproducibility below 5%. A set of nine hydrophobic and polar aromatic compounds (all noncharged) was used to investigate the retention mechanism. Possible candidates for chromatographic interaction and retention in these monoliths are the hydrophobic polymer backbone itself and the alkyl, carbonyl, hydroxy, amino, amide, and charged groups introduced by the various functional monomers. Judging from our results, the carbonyl and the hydroxy functions, as well as the hydrophobic polymer backbone can be supposed to be the main sites of interaction. The charged but also the alkyl functions seem to be less important in this regard. The polymerization conditions and especially the composition of the reaction mixture have a strong influence on the behavior of the final column.

Acrylamide↗

PubMed2002

Hyphenation of multi-dimensional chromatography and mass spectrometry for the at-line-analysis of the integrity of recombinant protein drugs.

A robust tool is proposed for the rapid at-line verification of the identity and integrity of (recombinant) proteins, namely the hyphenation of multidimensional chromatography and mass spectrometry (MS). A recombinant human antibody produced in Chinese hamster ovary cells is taken as pertinent example. The recombinant human antibody is first captured from the production environment by affinity chromatography (rProtein A, isolation/concentration of the target molecule) and automatically transferred to an enzyme reactor (immobilized trypsin column) for digestion, thereby yielding different peptides corresponding to the protein sequence. The peptides are then separated on a reversed-phase column before being analyzed and identified by MS. This step does not require a fine resolution since the mass spectrometer can identify a variety of substances at the same time. The results are then analyzed in silico with suitable bio-informatic tools. When the gene sequence of the protein product is known, proteolytic cleavages can be predicted and the exact mass and hence the amino acid sequence of each peptide can thereby be deduced. Fitting experimental data and reference peptide sequences then provides important information about the integrity of the protein and more particularly about its sequence. In our case, the integrity of 45% of the light and 75% of the heavy chain sequences of the antibody could be verified within minutes.

Amino Acid Sequence↗

PubMedDate not supplied

Continuous separation of multicomponent protein mixtures by annular displacement chromatography.

Displacement chromatography is a predominantly nonlinear mode of chromatography, which has certain advantages over the elution mode for preparative bioseparations. Whereas continuous production (and separation) processes have their theoretical benefits in this context, protein displacement chromatography has up to now only been performed in the batch mode. In this contribution, we demonstrate that the principle of continuous annular chromatography can be adapted to displacement chromatography. Separations of up to three standard proteins (two whey proteins, soybean trypsin inhibitor) were developed and optimized using a small (4 x 250 mm) batch column. These separations were subsequently transferred directly to the continuous system (500-mL column). Separations of similar quality in terms of final product purity and recovery yield were obtained using the continuous system.

Adsorption↗