Jeannie Horak | University of Tübingen (original) (raw)

Papers by Jeannie Horak

Journal of chromatography. A, Jan 2, 2016

A thin functional film of poly(3-mercaptopropyl)methylsiloxane was coated onto vinyl-modified sil... more A thin functional film of poly(3-mercaptopropyl)methylsiloxane was coated onto vinyl-modified silica particles (5μm, 100Å pore size) and chemically crosslinked to the surface. Excess of thiol functionalities allow bonding of alkene containing ligands by thiol-ene click reaction in a second step (QN-VII). Besides that a single step surface modification procedure was established in which alkene functional ligands were directly added to the polysiloxane coating solution and thus, after evaporation of the solvent, crosslinking to the vinylized surface and bonding of chromatographic ligand to the thiolated polysiloxane film occur simultaneously in one step (QN-VI). Successful bonding of the polysiloxane film was confirmed for both approaches by (29)Si cross-polarization/magic angle spinning NMR spectra. The new surface functionalization concept can be utilized as a new platform for the preparation of various low-bleed, mass spectrometry-compatible stationary phases with a variety of func...

Journal of Chromatography A, 2015

A series of new mixed-mode reversed-phase/weak anion-exchange (RP/WAX) phases have been synthesiz... more A series of new mixed-mode reversed-phase/weak anion-exchange (RP/WAX) phases have been synthesized by immobilization of N-undecenyl-3-α-aminotropane onto thiol-modified silica gel by thiol-ene click chemistry and subsequent introduction of acidic thiol-endcapping functionalities of different type and surface densities. Click chemistry allowed to adjust a controlled surface concentration of the RP/WAX ligand in such a way that a sufficient quantity of residual thiols remained unmodified which have been capped by thiol click with either 3-butenoic acid or allylsulfonic acid as co-ligands. In another embodiment, performic acid oxidation of N-undecenyl-3-α-aminotropane-derivatized thiol-modified silica gave a RP/WAX phase with high density of sulfonic acid end-capping groups. ζ-Potential determinations confirmed the fine-tuned pI of these mixed-mode stationary phases which was shifted from 9.5 to 8.2, 7.8, and 6.5 with 3-butenoic acid and allylsulfonic acid end-capping as well as performic acid oxidation. For acidic solutes, the co-ionic endcapping leads to strongly reduced retention times and clearly allowed elution of these analytes under lower ionic strength thus milder elution conditions. In spite of the acidic endcapping, the new mixed-mode phases maintained their hydrophobic and anion-exchange selectivity as well as their multimodal nature featuring RP and HILIC elution domains at acetonitrile percentages below and above 50%, respectively. Column classification by principal component analysis of an extended retention map in comparison to a set of polar commercial and in-house synthesized stationary phases confirmed complementarity of the new mixed-mode phases with respect to HILIC, polar RP, amino and commercial mixed-mode phases.

Food and Bioproducts Processing, 2013

The occurrence of d-amino acids in native and processed plant products is brought into context wi... more The occurrence of d-amino acids in native and processed plant products is brought into context with the harshness of their treatment condition. It was found that already a small increase in processing harshness such as the milling efficiency of wheat straw or the increased pressure and duration of pumpkin seed oil extraction leads to traceable changes in the overall amino acid content as well as the racemization rate of free and protein bound amino acids.

The cavitation field radiated by a 20 kHz sonotrode-type transducer is experimentally and theoret... more The cavitation field radiated by a 20 kHz sonotrode-type transducer is experimentally and theoretically analyzed. Special interest is paid to the origin of the strong fluid streaming appearing in low frequency sonoreactors. A new experimental procedure is proposed to evaluate the mean acoustic pressure inside the fluid. This parameter has been quantified for different points and amplitudes. The velocity of the radiating surface is controlled by a laser interferometer and is always sinusoidal. Train wave excitation is used. The pressure wave and amplitude are measured in the tank with a calibrated hydrophone. The acoustic mean pressure is estimated from the total pressure value at the end of the pulse after an adequate filtering. An analytical nonlinear second order model based on the coupling of the equations of the fluid mechanics with the Rayleigh-Plesset equation is developed in order to relate the measured acoustic parameters to the cavitation state of the fluid. The distributions of the fundamental amplitude and mean pressure are calculated as a function of bubble density and bubble size. A qualitative theoretical description of the experimental data is presented. Quantitative differences and model limitations are commented.

Rapid Communications in Mass Spectrometry, 2001

The plant growth regulator chlormequat, an involatile quaternary ammonium salt, has been quantifi... more The plant growth regulator chlormequat, an involatile quaternary ammonium salt, has been quantified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). Restrictions for quantitative MALDI-TOFMS analysis, such as irreproducible crystallisation and unsatisfactory laser stability, have been overcome by the application of two synthesised isotopically labelled standards and the optimisation of the measurement protocol. Data acquisition at constant laser power was compared to data acquisition at approximately constant ion abundance of the relevant ions (analyte and internal standards). Data acquisition at constant ion abundance performed better and enabled a high number of consecutive firings to the same sample deposition area. Furthermore an increased sample-to-sample repeatability and a high reproducibility over several weeks without re-calibration have been attained by this method. Linearity over three orders of magnitude (0.05 to 30 ng/microL chlormequat), with a correlation coefficient of 0.9997, was achieved using [13C3]-chlormequat as internal standard. Limit of detection and limit of determination were determined to be in the low pg/microL range for pure standard solutions. Thin-layer chromatography was applied for the removal of high amounts of choline, which is often present in plant tissue extracts and can adversely affect the ionisation and detection of chlormequat by MALDI-TOFMS. The use of two internal standards ([13C3]- and [2H9]-chlormequat) enabled direct quantification and simultaneous control of the recovery.

The occurrence of d-amino acids in native and processed plant products is brought into context wi... more The occurrence of d-amino acids in native and processed plant products is brought into context with the harshness of their treatment condition. It was found that already a small increase in processing harshness such as the milling efficiency of wheat straw or the increased pressure and duration of pumpkin seed oil extraction leads to traceable changes in the overall amino acid content as well as the racemization rate of free and protein bound amino acids.

Journal of Chromatography A, 2014

Synthetic oligonucleotides gain increasing importance in new therapeutic concepts and as probes i... more Synthetic oligonucleotides gain increasing importance in new therapeutic concepts and as probes in biological sciences. If pharmaceutical-grade purities are required, chromatographic purification using ion-pair reversed-phase chromatography is commonly carried out. However, separation selectivity for structurally closely related impurities is often insufficient, especially at high sample loads. In this study, a "mixed-mode" reversed-phase/weak anion exchanger stationary phase has been investigated as an alternative tool for chromatographic separation of synthetic oligonucleotides with minor sequence variations. The employed mixed-mode phase shows great flexibility in method development. It has been run in various gradient elution modes, viz. one, two or three parameter (mixed) gradients (altering buffer pH, buffer concentration, and organic modifier) to find optimal elution conditions and gain further insight into retention mechanisms. Compared to ion-pair reversed-phase and mere anion-exchange separation, enhanced selectivities were observed with the mixed-mode phase for 20-23 nucleotide (nt) long oligonucleotides with similar sequences. Oligonucleotides differing by 1, 2 or 3 nucleotides in length could be readily resolved and separation factors for single nucleotide replacements declined in the order Cytosine (C)/Guanine (G)>Adenine (A)/Guanine∼Guanine/Thymine (T)>Adenine/Cytosine∼Cytosine/Thymine>Adenine/Thymine. Selectivities were larger when the modification was at the 3' terminal-end, declined when it was in the middle of the sequence and was smallest when it was located at the 5' terminus. Due to the lower surface area of the 200Å pore size mixed-mode stationary phase compared to the corresponding 100Å material, lower retention times with equal selectivities under milder elution conditions were achievable. Considering high sample loading capacities of the mixed-mode anion-exchanger phase, it should have great potential for chromatographic oligonucleotide separation and purification.

Journal of Chromatography B, 2010

This report describes and compares different strategies to deactivate (endcap) epoxide groups and... more This report describes and compares different strategies to deactivate (endcap) epoxide groups and azide groups on bio-chromatographic support surfaces, before and after ligand attachment. Adsorbents possessing epoxide groups were deactivated using acidic hydrolysis or were endcapped with 2-mercaptoethanol or 2-ethanolamine. The influence of surface-bound 2-ethanolamine was demonstrated for the triazine-type affinity adsorbent B14-2LP-FractoAIMs-1, which was tested in combination with the weak anion exchange material 3-aminoquinuclidine-FractoAIMs-3 (AQ-FA3). Azide groups were modified with 2-propargylalcohol using Click-Chemistry. Besides the conventional one-pot Click reaction, an alternative approach was introduced. This optimized Click protocol was employed (i) for the preparation of the weak anion exchange material AdQ-triazole-Fractogel (AdQ-TRZ-FG) and (ii) for the endcapping of residual azide groups with 3-propargyl alcohol. Using the new Click reaction protocol the ligand immobilization rate was doubled from 250 to 500 μmol/g dry adsorbent. Furthermore, the modified support surface was proven to be inert towards the binding of immunoglobulin G (IgG) as well as feed impurities. A thorough evaluation of modified surfaces and adsorbents was performed with dynamic binding experiments using cell culture supernatant containing monoclonal human immunoglobulin G (h-IgG-1). Besides SDS-Page, a recently introduced Protein A-size exclusion HPLC method (PSEC-HPLC) was used to visualize the feed impurity composition and the IgG content of all collected sample fractions in simple PSEC-Plots. A surprising outcome of this study was the irreversible binding of IgG to azide modified surfaces. It was found that organic azide compounds, e.g. 1-azide-3-(2-propen-1-yloxy)-2-propanol (AGE-N3) promote antibody aggregation to a slightly higher extent than the inorganic sodium azide. The possibility that the Hofmeister Series of salt anions may be applicable to predict the properties of the corresponding organic compounds is discussed.

Journal of Chromatography A, 2008

Two novel sulfonyl-embedded reversed phase materials with sulfonic acid moieties (SO X -RP) were ... more Two novel sulfonyl-embedded reversed phase materials with sulfonic acid moieties (SO X -RP) were prepared by a simple oxidation of two silica-based sulfur-embedded RP-phases (S-RP). The chromatographic behavior of the resultant sulfonyl/sulfonic acid-embedded phase C3-SO 2 -C18/SO 3 H and the sulfonyl-embedded phase C3-SO 2 -C14 e.c. (silanol endcapped) was extensively elucidated with a number of well-established as well as self-assembled column tests. These SO X -RP phases were also compared with their corresponding S-RP phases as well as with two commercial carbonyl-containing RP phases (CO-RP) with amide-and urea-embedding. The SO X -RP phases were found to exhibit exceptionally high planar recognition ability for polyaromatic analytes at comparable retention times to the investigated CO-RP phases and highly reduced retention compared to the parent, non-oxidized S-RP-materials. The results of this study suggest that the selectivity enhancement observed with the S-oxidized phases is due to sulfonyl-and sulfonic acid-interactions. Furthermore these novel SO X -RP phases proved to be fully applicable under 100% aqueous mobile-phase conditions making them a useful alternative to conventional RP as well as polar-embedded CO-RP phases.

Journal of Chromatography A, 2010

The present work describes two HPLC-UV methods for multi-protein quantification using (i) only a ... more The present work describes two HPLC-UV methods for multi-protein quantification using (i) only a Protein A sensor cartridge (Protein A HPLC) and (ii) the same Protein A cartridge in combination with a size exclusion HPLC column (PSEC-HPLC). The possibility to simultaneously quantify immunoglobulin G (IgG) besides a non-binding protein such as bovine serum albumin (BSA) increases the applicability of Protein A HPLC. Its most pronounced feature is its independence of the buffer system, pH-value and salt content of the investigated sample solvent, which includes cell media. A comparison with the state-of-the-art, the photometrical Bradford method, shows that Protein A HPLC is as sensitive as Bradford, but that it comes with an extended linear range of 4 orders of magnitude, ranging from 0.15 [microg abs] to 1 [mg abs] absolute injected protein amount. The applicability of the PSEC-HPLC method is demonstrated for the analysis of real cell culture feed samples. While Protein A binds IgG, the SEC-column distributes the feed impurities by their molecular weight. The peak area ratios of IgG and the feed impurities of interest are then plotted against the collected sample fraction. These Protein A-Size-Exclusion-Chromatographic diagrams (PSEC-plot) combine the performance information of feed impurities and IgG in a single plot. Further it is shown that both methods are suitable for the performance evaluation of antibody purification media using static as well as dynamic binding experiments performed on DEAE-Fractogel and Capto Adhere. The investigated test samples were "mock" protein solutions with increasing complexity ranging from simple PBS buffer to serum free cell media and "real" cell culture feed solutions.

Journal of Chromatography A, 2004

The influence of sulfur-aromatic interactions on the chromatographic separation behavior of hybri... more The influence of sulfur-aromatic interactions on the chromatographic separation behavior of hybrid RP phases, containing thiol-groups and/or embedded sulfide-groups (S-RP) has been investigated. To allow a precise outline of this new interaction mode, a wide variety of S-RP phases with different alkyl chain length, with and without residual thiol-groups and silanol-endcapping were prepared and tested in comparison to some conventional monomerical as well as polymerical n-alkyl type RP phases. The solute test sets employed in this study comprised the classical chromatographic column tests from Engelhardt and Tanaka as well as test assemblies containing polycyclic aromatic hydrocarbons, stilbene-based cis/trans isomers and functional isomers of benzene. In general, a pronounced strong planar recognition ability as well as a strong increase in the retention for aromatic compounds have been noticed. It was furthermore found that not only the sulfur atom incorporated into the alkyl chain, but also the residual thiol-groups of the 3-propylthiol silica backbone contribute to the overall retention behavior of these novel S-RP type phases.

Journal of Chromatography A, 2011

The aim of this study was to investigate functional increments of ion exchange type ligands, whic... more The aim of this study was to investigate functional increments of ion exchange type ligands, which may improve the performance of mixed-modal ligands for antibody capture out of feed solutions with pH above 6.0 and containing sodium chloride concentrations of 150 mM and higher. For this purpose several functional groups such as sulfonyl, sulfanyl, amide, methoxy, short alkyl and aromatic moieties were tested in combination with a strong sulfonic acid and/or a weak carboxylic acid group. Therefore a series of ligands were synthesized and subsequently coupled onto epoxide activated Fractogel(®) EMD. In the first instance, all materials were tested by static binding capacity measurements (SBC) under test conditions, comprising a wide variety of different sodium chloride concentrations and differing pH values ranging from 4.5 to 7.5. From these preliminary experiment it was found that especially the aromatic groups improved the binding of human immunoglobulin G (h-IgG) under isotonic conditions, while other increments, e.g. thiophilic or amide groups, were not able to increase the capacity significantly. Taking the SBC results into account, the most promising materials were investigated under dynamic binding conditions (DBC) with a reduced selection of test conditions (pH 5.5, 6.5 and 7.4 at 75 and 150mM NaCl). N-benzoyl-homocysteine (material J) and 3,5-dimethoxybenzoyl-homocysteine (material K) showed 100% DBCs of 37 mg/mL and 32 mg/mL in the presence of 75 mM NaCl and pH 6.5. Material L carrying mercaptobenzoic acid as a ligand and tested with the same solution provided a 100% DBC of 68 mg/mL. The influence of Pluronic F68 in a mock feed solution as well as in cell culture supernatant was investigated with the best performing bio-affinity type adsorbent, material L. For the real sample feed subsequent SDS-PAGE was conducted for the collected fractions.

Food and Bioproducts Processing, 2013

The occurrence of d-amino acids in native and processed plant products is brought into context wi... more The occurrence of d-amino acids in native and processed plant products is brought into context with the harshness of their treatment condition. It was found that already a small increase in processing harshness such as the milling efficiency of wheat straw or the increased pressure and duration of pumpkin seed oil extraction leads to traceable changes in the overall amino acid content as well as the racemization rate of free and protein bound amino acids.

Analytical and Bioanalytical Chemistry, 2013

The fluorescent tag 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; AccQ Fluor reagent kit ... more The fluorescent tag 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; AccQ Fluor reagent kit from Waters) is a commercial N-terminal label for proteinogenic amino acids (AAs), designed for reversed-phase separation and quantification of the AA racemates. The applicability of AQC-tagged AAs and AA-type zwitterionic compounds was tested for enantiomer separation on the tert-butyl carbamate modified quinine and quinidine based chiral stationary phases, QN-AX and QD-AX employing polar-organic elution conditions. The investigated test analytes included the enantiomers of the positional isomers of isoleucine (Ile), threonine, homoserine, and 4-hydroxyproline. Furthermore, β-AAs, cyclic, and heterocyclic AAs including trans-2-amino-cyclohexane carboxylic acid and trans-2-aminocyclohexyl sulfonic acid, phenylalanine derivatives substituted with halides with increasing electronegativity and 3,4-dihydroxyphenylalanine, cysteine-related derivatives including homocysteic acid, methionine sulfone, cysteine-S-acetic acid, and cysteine-S-acetamide as well as a small range of aminophosphonic acids were enantioseparated. A mechanistic interaction study of AQC-AAs in comparison with fluoresceine isothiocyanate-labeled AAs was performed. The chiral and chemoselective recognition processes involved in enantiomer separation and retention was systematically discussed. Special emphasis was set on the influential factors exhibited by the chemistry, branching position, and spatial properties of the investigated zwitterionic analytes. The general interest to separate and distinguish between different types of branched-chained AAs and metabolic side products thereof lies in the toxicity of some of these compounds, which makes for instance allo-Ile an attractive candidate in disease-related biomarker research.

Analytical and Bioanalytical Chemistry, 2011

The new affinity-type Mimetic Ligand™ B14 was coupled with a 1,2-diaminoethane spacer (2LP) and a... more The new affinity-type Mimetic Ligand™ B14 was coupled with a 1,2-diaminoethane spacer (2LP) and a [1,2,3]-triazole spacer (TRZ) to three different support media. In addition to the agarose-based PuraBead and the polymethacrylate-type Fractogel, three new polymeric support media were introduced, the FractoAIMs 1, 2, and 3 (FA1, FA2, and FA3). These new FA supports differ in pore size as well as density of epoxide groups. The immobilization of the B14-ligand onto an azide-groupmodified surface was performed with a copper (I)-mediated Click reaction. The IgG capture performance was tested for various ligand-spacer support combinations using cell culture feed containing human immunoglobulin G 1 (hIgG 1 ). The most promising adsorbent, B14-TRZ-FA3, was further optimized by improving the surface chemistry through a triple endcapping concept employing an improved Click reaction protocol. This new technique enabled the most efficient deactivation of residual azide groups. In a direct comparison with a commercially available Protein A media, B14-TRZ-FA3 3× ec provided superior results at fast flow-rates and low bed-height. Dynamic binding capacities of 11.4 g/L for 10% breakthrough of hIgG 1 , elution capacities of 16.0 g/L hIgG 1 and a recovery of 86% were achieved. The same results were obtained for a dialyzed and pre-purified feed solution, which is a clear indicator that triple-endcapped affinity support surfaces are practically inert to the non-specific binding of host cell proteins.

Journal of chromatography. A, Jan 2, 2016

A thin functional film of poly(3-mercaptopropyl)methylsiloxane was coated onto vinyl-modified sil... more A thin functional film of poly(3-mercaptopropyl)methylsiloxane was coated onto vinyl-modified silica particles (5μm, 100Å pore size) and chemically crosslinked to the surface. Excess of thiol functionalities allow bonding of alkene containing ligands by thiol-ene click reaction in a second step (QN-VII). Besides that a single step surface modification procedure was established in which alkene functional ligands were directly added to the polysiloxane coating solution and thus, after evaporation of the solvent, crosslinking to the vinylized surface and bonding of chromatographic ligand to the thiolated polysiloxane film occur simultaneously in one step (QN-VI). Successful bonding of the polysiloxane film was confirmed for both approaches by (29)Si cross-polarization/magic angle spinning NMR spectra. The new surface functionalization concept can be utilized as a new platform for the preparation of various low-bleed, mass spectrometry-compatible stationary phases with a variety of func...

Journal of Chromatography A, 2015

A series of new mixed-mode reversed-phase/weak anion-exchange (RP/WAX) phases have been synthesiz... more A series of new mixed-mode reversed-phase/weak anion-exchange (RP/WAX) phases have been synthesized by immobilization of N-undecenyl-3-α-aminotropane onto thiol-modified silica gel by thiol-ene click chemistry and subsequent introduction of acidic thiol-endcapping functionalities of different type and surface densities. Click chemistry allowed to adjust a controlled surface concentration of the RP/WAX ligand in such a way that a sufficient quantity of residual thiols remained unmodified which have been capped by thiol click with either 3-butenoic acid or allylsulfonic acid as co-ligands. In another embodiment, performic acid oxidation of N-undecenyl-3-α-aminotropane-derivatized thiol-modified silica gave a RP/WAX phase with high density of sulfonic acid end-capping groups. ζ-Potential determinations confirmed the fine-tuned pI of these mixed-mode stationary phases which was shifted from 9.5 to 8.2, 7.8, and 6.5 with 3-butenoic acid and allylsulfonic acid end-capping as well as performic acid oxidation. For acidic solutes, the co-ionic endcapping leads to strongly reduced retention times and clearly allowed elution of these analytes under lower ionic strength thus milder elution conditions. In spite of the acidic endcapping, the new mixed-mode phases maintained their hydrophobic and anion-exchange selectivity as well as their multimodal nature featuring RP and HILIC elution domains at acetonitrile percentages below and above 50%, respectively. Column classification by principal component analysis of an extended retention map in comparison to a set of polar commercial and in-house synthesized stationary phases confirmed complementarity of the new mixed-mode phases with respect to HILIC, polar RP, amino and commercial mixed-mode phases.

Food and Bioproducts Processing, 2013

The occurrence of d-amino acids in native and processed plant products is brought into context wi... more The occurrence of d-amino acids in native and processed plant products is brought into context with the harshness of their treatment condition. It was found that already a small increase in processing harshness such as the milling efficiency of wheat straw or the increased pressure and duration of pumpkin seed oil extraction leads to traceable changes in the overall amino acid content as well as the racemization rate of free and protein bound amino acids.

The cavitation field radiated by a 20 kHz sonotrode-type transducer is experimentally and theoret... more The cavitation field radiated by a 20 kHz sonotrode-type transducer is experimentally and theoretically analyzed. Special interest is paid to the origin of the strong fluid streaming appearing in low frequency sonoreactors. A new experimental procedure is proposed to evaluate the mean acoustic pressure inside the fluid. This parameter has been quantified for different points and amplitudes. The velocity of the radiating surface is controlled by a laser interferometer and is always sinusoidal. Train wave excitation is used. The pressure wave and amplitude are measured in the tank with a calibrated hydrophone. The acoustic mean pressure is estimated from the total pressure value at the end of the pulse after an adequate filtering. An analytical nonlinear second order model based on the coupling of the equations of the fluid mechanics with the Rayleigh-Plesset equation is developed in order to relate the measured acoustic parameters to the cavitation state of the fluid. The distributions of the fundamental amplitude and mean pressure are calculated as a function of bubble density and bubble size. A qualitative theoretical description of the experimental data is presented. Quantitative differences and model limitations are commented.

Rapid Communications in Mass Spectrometry, 2001

The plant growth regulator chlormequat, an involatile quaternary ammonium salt, has been quantifi... more The plant growth regulator chlormequat, an involatile quaternary ammonium salt, has been quantified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). Restrictions for quantitative MALDI-TOFMS analysis, such as irreproducible crystallisation and unsatisfactory laser stability, have been overcome by the application of two synthesised isotopically labelled standards and the optimisation of the measurement protocol. Data acquisition at constant laser power was compared to data acquisition at approximately constant ion abundance of the relevant ions (analyte and internal standards). Data acquisition at constant ion abundance performed better and enabled a high number of consecutive firings to the same sample deposition area. Furthermore an increased sample-to-sample repeatability and a high reproducibility over several weeks without re-calibration have been attained by this method. Linearity over three orders of magnitude (0.05 to 30 ng/microL chlormequat), with a correlation coefficient of 0.9997, was achieved using [13C3]-chlormequat as internal standard. Limit of detection and limit of determination were determined to be in the low pg/microL range for pure standard solutions. Thin-layer chromatography was applied for the removal of high amounts of choline, which is often present in plant tissue extracts and can adversely affect the ionisation and detection of chlormequat by MALDI-TOFMS. The use of two internal standards ([13C3]- and [2H9]-chlormequat) enabled direct quantification and simultaneous control of the recovery.

The occurrence of d-amino acids in native and processed plant products is brought into context wi... more The occurrence of d-amino acids in native and processed plant products is brought into context with the harshness of their treatment condition. It was found that already a small increase in processing harshness such as the milling efficiency of wheat straw or the increased pressure and duration of pumpkin seed oil extraction leads to traceable changes in the overall amino acid content as well as the racemization rate of free and protein bound amino acids.

Journal of Chromatography A, 2014

Synthetic oligonucleotides gain increasing importance in new therapeutic concepts and as probes i... more Synthetic oligonucleotides gain increasing importance in new therapeutic concepts and as probes in biological sciences. If pharmaceutical-grade purities are required, chromatographic purification using ion-pair reversed-phase chromatography is commonly carried out. However, separation selectivity for structurally closely related impurities is often insufficient, especially at high sample loads. In this study, a "mixed-mode" reversed-phase/weak anion exchanger stationary phase has been investigated as an alternative tool for chromatographic separation of synthetic oligonucleotides with minor sequence variations. The employed mixed-mode phase shows great flexibility in method development. It has been run in various gradient elution modes, viz. one, two or three parameter (mixed) gradients (altering buffer pH, buffer concentration, and organic modifier) to find optimal elution conditions and gain further insight into retention mechanisms. Compared to ion-pair reversed-phase and mere anion-exchange separation, enhanced selectivities were observed with the mixed-mode phase for 20-23 nucleotide (nt) long oligonucleotides with similar sequences. Oligonucleotides differing by 1, 2 or 3 nucleotides in length could be readily resolved and separation factors for single nucleotide replacements declined in the order Cytosine (C)/Guanine (G)>Adenine (A)/Guanine∼Guanine/Thymine (T)>Adenine/Cytosine∼Cytosine/Thymine>Adenine/Thymine. Selectivities were larger when the modification was at the 3' terminal-end, declined when it was in the middle of the sequence and was smallest when it was located at the 5' terminus. Due to the lower surface area of the 200Å pore size mixed-mode stationary phase compared to the corresponding 100Å material, lower retention times with equal selectivities under milder elution conditions were achievable. Considering high sample loading capacities of the mixed-mode anion-exchanger phase, it should have great potential for chromatographic oligonucleotide separation and purification.

Journal of Chromatography B, 2010

This report describes and compares different strategies to deactivate (endcap) epoxide groups and... more This report describes and compares different strategies to deactivate (endcap) epoxide groups and azide groups on bio-chromatographic support surfaces, before and after ligand attachment. Adsorbents possessing epoxide groups were deactivated using acidic hydrolysis or were endcapped with 2-mercaptoethanol or 2-ethanolamine. The influence of surface-bound 2-ethanolamine was demonstrated for the triazine-type affinity adsorbent B14-2LP-FractoAIMs-1, which was tested in combination with the weak anion exchange material 3-aminoquinuclidine-FractoAIMs-3 (AQ-FA3). Azide groups were modified with 2-propargylalcohol using Click-Chemistry. Besides the conventional one-pot Click reaction, an alternative approach was introduced. This optimized Click protocol was employed (i) for the preparation of the weak anion exchange material AdQ-triazole-Fractogel (AdQ-TRZ-FG) and (ii) for the endcapping of residual azide groups with 3-propargyl alcohol. Using the new Click reaction protocol the ligand immobilization rate was doubled from 250 to 500 μmol/g dry adsorbent. Furthermore, the modified support surface was proven to be inert towards the binding of immunoglobulin G (IgG) as well as feed impurities. A thorough evaluation of modified surfaces and adsorbents was performed with dynamic binding experiments using cell culture supernatant containing monoclonal human immunoglobulin G (h-IgG-1). Besides SDS-Page, a recently introduced Protein A-size exclusion HPLC method (PSEC-HPLC) was used to visualize the feed impurity composition and the IgG content of all collected sample fractions in simple PSEC-Plots. A surprising outcome of this study was the irreversible binding of IgG to azide modified surfaces. It was found that organic azide compounds, e.g. 1-azide-3-(2-propen-1-yloxy)-2-propanol (AGE-N3) promote antibody aggregation to a slightly higher extent than the inorganic sodium azide. The possibility that the Hofmeister Series of salt anions may be applicable to predict the properties of the corresponding organic compounds is discussed.

Journal of Chromatography A, 2008

Two novel sulfonyl-embedded reversed phase materials with sulfonic acid moieties (SO X -RP) were ... more Two novel sulfonyl-embedded reversed phase materials with sulfonic acid moieties (SO X -RP) were prepared by a simple oxidation of two silica-based sulfur-embedded RP-phases (S-RP). The chromatographic behavior of the resultant sulfonyl/sulfonic acid-embedded phase C3-SO 2 -C18/SO 3 H and the sulfonyl-embedded phase C3-SO 2 -C14 e.c. (silanol endcapped) was extensively elucidated with a number of well-established as well as self-assembled column tests. These SO X -RP phases were also compared with their corresponding S-RP phases as well as with two commercial carbonyl-containing RP phases (CO-RP) with amide-and urea-embedding. The SO X -RP phases were found to exhibit exceptionally high planar recognition ability for polyaromatic analytes at comparable retention times to the investigated CO-RP phases and highly reduced retention compared to the parent, non-oxidized S-RP-materials. The results of this study suggest that the selectivity enhancement observed with the S-oxidized phases is due to sulfonyl-and sulfonic acid-interactions. Furthermore these novel SO X -RP phases proved to be fully applicable under 100% aqueous mobile-phase conditions making them a useful alternative to conventional RP as well as polar-embedded CO-RP phases.

Journal of Chromatography A, 2010

The present work describes two HPLC-UV methods for multi-protein quantification using (i) only a ... more The present work describes two HPLC-UV methods for multi-protein quantification using (i) only a Protein A sensor cartridge (Protein A HPLC) and (ii) the same Protein A cartridge in combination with a size exclusion HPLC column (PSEC-HPLC). The possibility to simultaneously quantify immunoglobulin G (IgG) besides a non-binding protein such as bovine serum albumin (BSA) increases the applicability of Protein A HPLC. Its most pronounced feature is its independence of the buffer system, pH-value and salt content of the investigated sample solvent, which includes cell media. A comparison with the state-of-the-art, the photometrical Bradford method, shows that Protein A HPLC is as sensitive as Bradford, but that it comes with an extended linear range of 4 orders of magnitude, ranging from 0.15 [microg abs] to 1 [mg abs] absolute injected protein amount. The applicability of the PSEC-HPLC method is demonstrated for the analysis of real cell culture feed samples. While Protein A binds IgG, the SEC-column distributes the feed impurities by their molecular weight. The peak area ratios of IgG and the feed impurities of interest are then plotted against the collected sample fraction. These Protein A-Size-Exclusion-Chromatographic diagrams (PSEC-plot) combine the performance information of feed impurities and IgG in a single plot. Further it is shown that both methods are suitable for the performance evaluation of antibody purification media using static as well as dynamic binding experiments performed on DEAE-Fractogel and Capto Adhere. The investigated test samples were "mock" protein solutions with increasing complexity ranging from simple PBS buffer to serum free cell media and "real" cell culture feed solutions.

Journal of Chromatography A, 2004

The influence of sulfur-aromatic interactions on the chromatographic separation behavior of hybri... more The influence of sulfur-aromatic interactions on the chromatographic separation behavior of hybrid RP phases, containing thiol-groups and/or embedded sulfide-groups (S-RP) has been investigated. To allow a precise outline of this new interaction mode, a wide variety of S-RP phases with different alkyl chain length, with and without residual thiol-groups and silanol-endcapping were prepared and tested in comparison to some conventional monomerical as well as polymerical n-alkyl type RP phases. The solute test sets employed in this study comprised the classical chromatographic column tests from Engelhardt and Tanaka as well as test assemblies containing polycyclic aromatic hydrocarbons, stilbene-based cis/trans isomers and functional isomers of benzene. In general, a pronounced strong planar recognition ability as well as a strong increase in the retention for aromatic compounds have been noticed. It was furthermore found that not only the sulfur atom incorporated into the alkyl chain, but also the residual thiol-groups of the 3-propylthiol silica backbone contribute to the overall retention behavior of these novel S-RP type phases.

Journal of Chromatography A, 2011

The aim of this study was to investigate functional increments of ion exchange type ligands, whic... more The aim of this study was to investigate functional increments of ion exchange type ligands, which may improve the performance of mixed-modal ligands for antibody capture out of feed solutions with pH above 6.0 and containing sodium chloride concentrations of 150 mM and higher. For this purpose several functional groups such as sulfonyl, sulfanyl, amide, methoxy, short alkyl and aromatic moieties were tested in combination with a strong sulfonic acid and/or a weak carboxylic acid group. Therefore a series of ligands were synthesized and subsequently coupled onto epoxide activated Fractogel(®) EMD. In the first instance, all materials were tested by static binding capacity measurements (SBC) under test conditions, comprising a wide variety of different sodium chloride concentrations and differing pH values ranging from 4.5 to 7.5. From these preliminary experiment it was found that especially the aromatic groups improved the binding of human immunoglobulin G (h-IgG) under isotonic conditions, while other increments, e.g. thiophilic or amide groups, were not able to increase the capacity significantly. Taking the SBC results into account, the most promising materials were investigated under dynamic binding conditions (DBC) with a reduced selection of test conditions (pH 5.5, 6.5 and 7.4 at 75 and 150mM NaCl). N-benzoyl-homocysteine (material J) and 3,5-dimethoxybenzoyl-homocysteine (material K) showed 100% DBCs of 37 mg/mL and 32 mg/mL in the presence of 75 mM NaCl and pH 6.5. Material L carrying mercaptobenzoic acid as a ligand and tested with the same solution provided a 100% DBC of 68 mg/mL. The influence of Pluronic F68 in a mock feed solution as well as in cell culture supernatant was investigated with the best performing bio-affinity type adsorbent, material L. For the real sample feed subsequent SDS-PAGE was conducted for the collected fractions.

Food and Bioproducts Processing, 2013

The occurrence of d-amino acids in native and processed plant products is brought into context wi... more The occurrence of d-amino acids in native and processed plant products is brought into context with the harshness of their treatment condition. It was found that already a small increase in processing harshness such as the milling efficiency of wheat straw or the increased pressure and duration of pumpkin seed oil extraction leads to traceable changes in the overall amino acid content as well as the racemization rate of free and protein bound amino acids.

Analytical and Bioanalytical Chemistry, 2013

The fluorescent tag 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; AccQ Fluor reagent kit ... more The fluorescent tag 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; AccQ Fluor reagent kit from Waters) is a commercial N-terminal label for proteinogenic amino acids (AAs), designed for reversed-phase separation and quantification of the AA racemates. The applicability of AQC-tagged AAs and AA-type zwitterionic compounds was tested for enantiomer separation on the tert-butyl carbamate modified quinine and quinidine based chiral stationary phases, QN-AX and QD-AX employing polar-organic elution conditions. The investigated test analytes included the enantiomers of the positional isomers of isoleucine (Ile), threonine, homoserine, and 4-hydroxyproline. Furthermore, β-AAs, cyclic, and heterocyclic AAs including trans-2-amino-cyclohexane carboxylic acid and trans-2-aminocyclohexyl sulfonic acid, phenylalanine derivatives substituted with halides with increasing electronegativity and 3,4-dihydroxyphenylalanine, cysteine-related derivatives including homocysteic acid, methionine sulfone, cysteine-S-acetic acid, and cysteine-S-acetamide as well as a small range of aminophosphonic acids were enantioseparated. A mechanistic interaction study of AQC-AAs in comparison with fluoresceine isothiocyanate-labeled AAs was performed. The chiral and chemoselective recognition processes involved in enantiomer separation and retention was systematically discussed. Special emphasis was set on the influential factors exhibited by the chemistry, branching position, and spatial properties of the investigated zwitterionic analytes. The general interest to separate and distinguish between different types of branched-chained AAs and metabolic side products thereof lies in the toxicity of some of these compounds, which makes for instance allo-Ile an attractive candidate in disease-related biomarker research.

Analytical and Bioanalytical Chemistry, 2011

The new affinity-type Mimetic Ligand™ B14 was coupled with a 1,2-diaminoethane spacer (2LP) and a... more The new affinity-type Mimetic Ligand™ B14 was coupled with a 1,2-diaminoethane spacer (2LP) and a [1,2,3]-triazole spacer (TRZ) to three different support media. In addition to the agarose-based PuraBead and the polymethacrylate-type Fractogel, three new polymeric support media were introduced, the FractoAIMs 1, 2, and 3 (FA1, FA2, and FA3). These new FA supports differ in pore size as well as density of epoxide groups. The immobilization of the B14-ligand onto an azide-groupmodified surface was performed with a copper (I)-mediated Click reaction. The IgG capture performance was tested for various ligand-spacer support combinations using cell culture feed containing human immunoglobulin G 1 (hIgG 1 ). The most promising adsorbent, B14-TRZ-FA3, was further optimized by improving the surface chemistry through a triple endcapping concept employing an improved Click reaction protocol. This new technique enabled the most efficient deactivation of residual azide groups. In a direct comparison with a commercially available Protein A media, B14-TRZ-FA3 3× ec provided superior results at fast flow-rates and low bed-height. Dynamic binding capacities of 11.4 g/L for 10% breakthrough of hIgG 1 , elution capacities of 16.0 g/L hIgG 1 and a recovery of 86% were achieved. The same results were obtained for a dialyzed and pre-purified feed solution, which is a clear indicator that triple-endcapped affinity support surfaces are practically inert to the non-specific binding of host cell proteins.