Salt Concentration In Mobile Phase Research Articles

Mechanistic modeling of empty-full separation in recombinant adeno-associated virus production using anion-exchange membrane chromatography.

Recombinant adeno-associated viral vectors (rAAVs) have become an industry-standard technology in the field of gene therapy, but there are still challenges to be addressed in their biomanufacturing. One of the biggest challenges is the removal of capsid species other than that which contains the gene of interest. In this work, we develop a mechanistic model for the removal of empty capsids-those that contain no genetic material-and enrichment of full rAAV using anion-exchange membrane chromatography. The mechanistic model was calibrated using linear gradient experiments, resulting in good agreement with the experimental data. The model was then applied to optimize the purification process through maximization of yield studying the impact of mobile phase salt concentration and pH, isocratic wash and elution length, flow rate, percent full (purity) requirement, loading density (challenge), and the use of single-step or two-step elution modes. A solution from the optimization with purity of 90% and recovery yield of 84% was selected and successfully validated, as the model could predict the recovery yield with remarkable fidelity and was able to find process conditions that led to significant enrichment. This is, to the best of our knowledge, the first case study of the application of de novo mechanistic modeling for the enrichment of full capsids in rAAV manufacturing, and it serves as demonstration of the potential of mechanistic modeling in rAAV process development.

Assessment of automated off-line solid-phase extraction LC-MS/MS to monitor EPA priority endocrine disruptors in tap water, surface water, and wastewater.

EPA method 539.1 recently introduced an expanded list of priority endocrine-disrupting compounds (EDCs), some of which were also included in the Unregulated Contaminant Monitoring Rule 3 (UCMR3). Though standardized methods are available for drinking water, analysis of steroid hormones and bisphenol A (BPA) at the ultra-trace level remains challenging. This study set out to evaluate the suitability of automated off-line solid-phase extraction (SPE) liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) for the determination of EPA-priority EDCs in environmental water matrixes (tap water, surface water, and wastewater influents and effluents). The target molecules included 14 steroid hormones (altrenogest, androstenedione, equilenin, equilin, α-estradiol, β-estradiol, estriol, estrone, ethinylestradiol, levonorgestrel, medroxyprogesterone, norethindrone, progesterone, testosterone) and BPA. Factors that may influence the analytical performance were assessed. This involved, for instance, testing combinations of SPE materials from different brands and protocol variations. Several materials presented absolute extraction efficiencies in acceptable ranges. Initial sample pH, nature of reconstitution medium, and mobile phase salt concentration were among the potential factors affecting analyte signal. Storage conditions (different preservative agents) possibly exerted the strongest influence, in agreement with the literature. Limits of detection were in the range of 0.03-0.5ng/L in drinking water, 0.1-0.5ng/L in surface water, and 0.16-1ng/L in wastewater. Method validation also involved testing linearity, accuracy, and precision in reagent water and matrix-matched extracted calibrants. The method was applied to field-collected water samples in Eastern Canada. Summed EDC concentrations remained low in tap water (<LOQ-0.92ng/L), while higher detection frequencies and contamination levels were reported in riverine surface waters (2.6-37ng/L) and municipal wastewaters (10-424ng/L).

Systematic Development of a Size Exclusion Chromatography Method for a Monoclonal Antibody with High Surface Aggregation Propensity (SAP) Index.

Size Exclusion Chromatography (SEC) has been widely used to assess aggregate content in bio-pharmaceutical drugs such as monoclonal antibodies (mAbs), and is routinely used during method development and release testing. Electrostatic interactions between protein analytes and SEC column resin are commonly observed besides the primary mode of size separation during SEC method development, which needs to be minimized. An effective method to minimize electrostatic interactions is through increasing mobile phase (MP) salt concentration. However; increasing salt concentration in MP will induce increased hydrophobicity of proteins and increased hydrophobic interactions between protein and stationary phase, as demonstrated for mAb-A in this paper, a protein with high surface aggregation propensity (SAP) score and an isoelectric point near mobile phase pH. In this work, a systematic, Design of Experimental approach was taken to identify optimal SEC method conditions including column type, buffer composition, ionic strength, pH and additives. The optimized method was demonstrated to be robust towards small changes in method operation conditions and was qualified for use in product release and stability studies. Additionally, biophysical and computational studies were performed to elucidate the role of MP additives, which supports the use of arginine as an essential additive to minimize undesirable hydrophobic interactions between proteins and stationary phase.

Impact of salt concentration in mobile phase on antibody retention in Protein A, Protein L and KappaSelect affinity chromatography

Protein A, Protein L and KappaSelect affinity resins have been widely used for antibody purification. Elution of antibody bound to these resins is typically achieved by acidic pH. In addition, elution can be moderately adjusted by tuning the salt concentration in mobile phase as hydrophobic interactions play a major role in binding. In this study, we assessed the impact of salt concentration in mobile phase on antibody retention in these three types of affinity chromatography. The data suggest that salt concentration has a bigger impact on retention in the two light chain-binding affinity columns (i.e., Protein L and KappaSelect) than in Protein A column. In particular, lowering salt concentration in mobile phase for Protein L and KappaSelect columns allows elution become feasible at higher pH. In addition, this finding suggests that wash in these two types of column aimed at removing weakly-bound byproducts can also be performed at increased pH by lowering salt concentration in the wash buffer. Rendering wash and elution feasible at higher pH has practical value for cases where the target antibodies are sensitive to stringent conditions.

Hybrid mode of hydrophobic interaction chromatography of monoclonal antibodies and related biomolecules: Influence of elution conditions on chromatographic performance using poly (alkyl aspartamide) silica columns

A hybrid mode of hydrophobic interaction chromatography (HHIC) is an emerging analytical technique for the separation of biomolecules under non-denaturing conditions that combines elements of conventional hydrophobic interaction and reversed-phase chromatography. This article explores the impact of mobile phase composition such as salt concentration and organic modifier on the separation of therapeutic monoclonal antibodies and related large biomolecules using poly (alkyl aspartamide) silica HIC columns. The initial mobile phase salt concentration had a significant impact on the separation of a mixture of large biomolecules demonstrating that the relationship of elution and salt concentration was more complex than in conventional HIC. In general, the earlier eluting components exhibited greater retention at higher salt concentration as is typical of HIC separations. Conversely, the later eluting components showed greater retention at lower initial salt concentration. This differential is useful for improving the overall separation by widening the elution window for components of a mixture. In addition, no significant unfolding of the proteins was detected by intrinsic fluorescence or electrospray mass spectrometry. The impact of linear velocity and gradient steepness was also evaluated.

On-line Fast Purification of Three Kinds of Active Proteins from Egg White with Two-dimensional Liquid Chromatography Using a Single Column

With the development of biotechnology and life science, both recombinant protein drug productions and proteomics research depend largely on fast and efficient protein separation technology. Analysis of complex samples has put forward higher and higher requirements to separation science. Therefore, developing new separation material, separation mode and more sensitive detection method should be one of the effective ways to solve these problems. Based on a novel strong cation exchange/hydrophobic interaction chromatography (SCX/HIC) dual-function column, which displays HIC character in a high salt concentration, and IEC character in a low salt concentration in mobile phase employed and can be employed to separate proteins with SCX and HIC modes, respectively, a new on-line two-dimensional liquid chromatography technology with a single column (2DLC-1C) was developed. An equipment with automatic control system called as "ChromatoExpert" for pro- cessing fast protein separation online 2DLC-1C was constructed, which has multiple functions, such as on-line fraction col- lection from the first separation mode and quantitatively sample re-injection into the second separation mode, on-line buffer exchange, etc. Eight kinds of protein standards were separated using SCX/HIC 2DLC-1C. The unretained and uncompletely separated proteins in SCX mode were collected online and could be separated completely with each other by online re-injection and second dimensional separation of HIC mode. As a result, these eight kinds of proteins can be separated with- in 2 h. It also was applied to purify three kinds of active intact proteins from egg white successfully. The results indicated that lysozyme, ovotransferrin and ovalbumin from egg white could be purified just within 70 min by online 2DLC-1C. The purity of these proteins obtained was 95%, 93% and 97%, respectively. The new online 2DLC-1C technology can simplify the puri- fication process and lower the separation cost. It is easy to realize automation and scale-up to preparative scale. Therefore, this new technology has a broad application prospect in the fast separation and preparation of active protein. Keywords online 2DLC-1C; protein; purification; egg white

Evaluation of retention behavior of carbohydrate compounds on the zwitterionic hydrophilic interaction liquid chromatographic column

Because of the strong polarity of carbohydrate compounds which have weak retention in reverse-phase liquid chromatography (RPLC) , hydrophilic interaction liquid chromatography (HILIC) is suitable for the separation and analysis of the carbohydrate compounds. In this study, nine carbohydrate compounds were selected as the test probes to systematically evaluate the retention behavior of the carbohydrate compounds on a Click TE-Cys column with zwitterionic stationary phase. And the effects of the ratio of organic phase and salt concentration in mobile phase on the retention behavior were investigated as well. The experiments proved that the nine carbohydrate compounds could be eluted in the order of their polarities from the Click TE-Cys column. With the increase of organic phase ratio, the retention of carbohydrate compounds was enhanced. As the increase of salt concentration, the retention of the carbohydrate compounds increased except sialic acid. Using displacement-adsorption liquid phase interaction model to simulate the retention behavior of the carbohydrate compounds under HILIC, the retention behavior of HILIC was described by the retention equation of ln k = a + bln C(B) + cC(B), and the retention values of HILIC were analyzed by multiple linear regression. The results demonstrated that the retention behavior of carbohydrate compounds on the Click TE-Cys was in accordance with the retention regularity of HILIC.

High ionic strength narrows the population of sites participating in protein ion-exchange adsorption: A single-molecule study

The retention and elution of proteins in ion-exchange chromatography is routinely controlled by adjusting the mobile phase salt concentration. It has repeatedly been observed, as judged from adsorption isotherms, that the apparent heterogeneity of adsorption is lower at more-eluting, higher ionic strength. Here, we present an investigation into the mechanism of this phenomenon using a single-molecule, super-resolution imaging technique called motion-blur Points Accumulation for Imaging in Nanoscale Topography (mbPAINT). We observed that the number of functional adsorption sites was smaller at high ionic strength and that these sites had reduced desorption kinetic heterogeneity, and thus narrower predicted elution profiles, for the anion-exchange adsorption of α-lactalbumin on an agarose-supported, clustered-charge ligand stationary phase. Explanations for the narrowing of the functional population such as inter-protein interactions and protein or support structural changes were investigated through kinetic analysis, circular dichroism spectroscopy, and microscopy of agarose microbeads, respectively. The results suggest the reduction of heterogeneity is due to both electrostatic screening between the protein and ligand and tuning the steric availability within the agarose support. Overall, we have shown that single molecule spectroscopy can aid in understanding the influence of ionic strength on the population of functional adsorbent sites participating in the ion-exchange chromatographic separation of proteins.

Development and evaluation of new imidazolium-based zwitterionic stationary phases for hydrophilic interaction chromatography

Hydrophilic interaction liquid chromatography (HILIC) has been widely used for separating polar compounds as a complement mode to reversed-phase liquid chromatography. The development of new stationary phases for HILIC is significant to improve the coverage of various polar and hydrophilic compounds. The present study described the preparation and application of novel imidazolium-based zwitterionic stationary phases. 1-Vinyl-3-(butyl-4-sulfonate) imidazolium was synthesized from 1-vinylimidazole and 1,4-butane sultone, then bonded to the surface of 3-mercaptopropyl modified silica particles (core–shell silica and totally porous silica) by “thiol-ene” click chemistry to obtain the resulting zwitterionic stationary phase with a positively charged imidazole ring and a negatively charged sulfonate group. The zwitterionic stationary phases exhibited good selectivity and favorable retention for a wide range of polar solutes (nucleosides, nucleic acid bases, benzoic organic acids, uric acid and its methyl derivatives, water-soluble vitamins) as compared to a bare silica column. The column efficiency could reach up to 100,000 theoretical plates/m with cytosine as the test solute. The retention changes of various types of test solutes were investigated under different chromatographic conditions including water content, pH, buffer salt concentration in mobile phase and column temperature. The results indicated that the retention of solutes on the stationary phase was the outcome of a mixed-mode retention mechanism (i.e. a combination of adsorptive and partitioning interactions). In conclusion, the new imidazolium-based zwitterionic stationary phases have shown excellent chromatographic behavior for a variety of polar solutes under HILIC mode, and have a great potential as a new type of stationary phases for HILIC.

Preparation of a novel dual-function strong cation exchange/hydrophobic interaction chromatography stationary phase for protein separation

We have explored a novel dual-function stationary phase which combines both strong cation exchange (SCX) and hydrophobic interaction chromatography (HIC) characteristics. The novel dual-function stationary phase is based on porous and spherical silica gel functionalized with ligand containing sulfonic and benzyl groups capable of electrostatic and hydrophobic interaction functionalities, which displays HIC character in a high salt concentration, and IEC character in a low salt concentration in mobile phase employed. As a result, it can be employed to separate proteins with SCX and HIC modes, respectively. The resolution and selectivity of the dual-function stationary phase were evaluated under both HIC and SCX modes with standard proteins and can be comparable to that of conventional IEC and HIC columns. More than 96% of mass and bioactivity recoveries of proteins can be achieved in both HIC and SCX modes, respectively. The results indicated that the novel dual-function column could replace two individual SCX and HIC columns for protein separation. Mixed retention mechanism of proteins on this dual-function column based on stoichiometric displacement theory (SDT) in LC was investigated to find the optimal balance of the magnitude of electrostatic and hydrophobic interactions between protein and the ligand on the silica surface in order to obtain high resolution and selectivity for protein separation. In addition, the effects of the hydrophobicity of the ligand of the dual-function packings and pH of the mobile phase used on protein separation were also investigated in detail. The results show that the ligand with suitable hydrophobicity to match the electrostatic interaction is very important to prepare the dual-function stationary phase, and a better resolution and selectivity can be obtained at pH 6.5 in SCX mode. Therefore, the dual-function column can replace two individual SCX and HIC columns for protein separation and be used to set up two-dimensional liquid chromatography with a single column (2DLC-1C), which can also be employed to separate three kinds of active proteins completely, such as lysozyme, ovotransferrin and ovalbumin from egg white. The result is very important not only to the development of new 2DLC technology with a single column for proteomics, but also to recombinant protein drug production for saving column expense and simplifying the process in biotechnology.

Refolding of lysozyme in hydrophobic interaction chromatography: Effects of hydrophobicity of adsorbent and salt concentration in mobile phase

The effects of salt concentration in mobile phase, elution strategy, and hydrophobicity of stationary phase on lysozyme refolding in hydrophobic interaction chromatography (HIC) were investigated. Butyl Sepharose 4 Fast Flow, the least hydrophobic HIC resin among the tested adsorbent, showed the best refolding yield. The binding efficiency of unfolded lysozyme on the adsorbent was maximized when 1 and 0.4 M of initial and final concentration of ammonium sulfate was used in mobile phase, respectively. The optimum gradient strategy for refolding and elution of lysozyme was determined as linear increase of urea concentration to 4M. The optimized condition suggests the less hydrophobic environment than conventionally used salt solutions and HIC resins. Consequently, total refolding yield was improved 1.6 times comparing with optimized dilution-based batch refolding method.

A HILIC method for the analysis of tromethamine as the counter ion in an investigational pharmaceutical salt

A hydrophilic interaction chromatography (HILIC) method using an aminopropyl stationary phase was developed and validated to determine the content of tromethamine as counter ion in an investigational drug substance. Tromethamine was a very polar compound without any chromophores, and could not be readily retained and detected by conventional reserved-phase HPLC-UV methods. Furthermore, the tromethamine salt of the drug compound also had limited solubility in aqueous solution. The method employed simple acetonitrile/water mobile phase (80/20, v/v) to provide sufficient retention for tromethamine. Meanwhile, the high acetonitrile content also helped to dissolve the drug substance sample. Refractive index (RI) was used for the detection of tromethamine. The method was found to be specific without interference from the drug compound and related impurities. The method was also validated for suitable precision, linearity and accuracy for the analysis of drug substance samples. The effects of various parameters, such as acetonitrile content, mobile phase salt concentration, and column temperature on HILIC separation were investigated.

Characterization and modeling of monolithic stationary phases: application to preparative chromatography

A methodology for characterizing and modeling preparative separations on monolithic ion-exchange stationary phases is presented. A dimensionless group analysis was carried out to determine the relative importance of mass transfer and kinetic resistances on this stationary phase. In contrast to conventional beaded morphologies, the continuous bed stationary phase was found to possess enhanced mass transport properties resulting in kinetic resistance as the dominant non-ideality. Accordingly, a reaction-dispersive steric-mass action formalism was successfully utilized for simulating preparative displacement chromatography on this resin. Since kinetics were found to be important on this column morphology, mobile phase salt concentration was found to be an important variable during displacement chromatography on this stationary phase. An increase in the mobile phase salt concentration was found to significantly improve the displacement separation of a model protein mixture. The formalism presented in this paper provides a better understanding of preparative chromatography in monolithic resin systems and a means of simulating separations on this class of chromatographic stationary phases.

Investigation of displacer equilibrium properties and mobile phase operating conditions in ion-exchange displacement chromatography

One of the major advantages of displacement chromatography is the simultaneous concentration and purification that can be effected during the process. The steric mass-action model of non-linear ion-exchange chromatography is employed to investigate the affects of mobile phase salt concentration and displacer equilibrium properties on the concentration of proteins in isotachic displacement zones. The results indicate that the salt microenvironment in displacement zones plays a major role in determining the concentration of the displaced proteins. The parameters which affect the salt microenvironment are both the initial salt concentration in the carrier as well as the induced gradient produced during the displacement process. For a given breakthrough volume, the induced gradient is determined by the ratio of the steric factor to the characteristic charge of the displacer. These results indicate that the use of relatively low salt concentrations in the carrier along with displacers with relatively high steric factor to characteristic charge ratios will produce significant concentration of the proteins during the purification process.

Salt effects in anion exchange displacement chromatography: Comparison of pentosan polysulfate and dextran sulfate displacers

Experimental studies were carried out to investigate the utility of pentosan polysulfate as a low molecular weight polyelectrolyte displacer for the purification of proteins in anion-exchange displacement systems. In addition, the influence of mobile phase salt concentration on displacer efficacy, protein-protein resolution, and displacement development were studied for several anionic displacers. It was found that while large polyelectrolytes (50 kd dextran sulfate) were efficient displacers for a wide range of salt concentrations, relatively small polyelectrolytes (3 kd pentosan polysulfate) were seen to act as an efficient displacer only under conditions of high salt micro-environments. In addition, for proteins exhibiting similar affinities, zone mixing at the protein-protein boundary was found to be quite sensitive to the salt concentration. Finally, displacement chromatography was successfully implemented for the separation of proteins from milk whey.