Linear Gradient Chromatography Research Articles

Demonstration of continuous gradient elution functionality with automated liquid handling systems for high-throughput purification process development

Various high-throughput systems and strategies are employed by the biopharmaceutical industry for early to late-stage process development for biologics manufacturing. The associated increases to experiment productivity and reduction in material consumption makes high throughput tools integral for bioprocess development. While these high-throughput systems have been successfully leveraged to generate high quality data representative of manufacturing scale processes, their data interpretation often requires complex data transformation and time-intensive system characterization. With respect to high throughput purification development, RoboColumns by Repligen operated on Tecan automated liquid handling systems offer superior performance scalability, but lack an optimized liquid delivery system that is representative of preparative chromatography. Particularly, stock Tecan liquid handling systems lack the capability to provide high-capacity continuous liquid flow and ideal linear gradient chromatography conditions. These limitations impact protein chromatography performance and hinder the application of high-throughput gradient elution experiments. In this work, we describe a Tecan Freedom EVO high-throughput purification tool that provides more continuous liquid delivery enabling continuous gradient elution capability for RoboColumn experiments as demonstrated by generation of highly linear conductivity gradients. Results demonstrate that the tool can provide RoboColumn performance and product quality data that is in agreement with larger, bench scale chromatography formats for two model purification methods. The described gradient purification method also provides more consistent performance between RoboColumns and larger column formats compared to step elution methods using the same optimized Tecan system. Lastly, new insights into the impact of discontinuous flow on RoboColumn elution performance are introduced, which may help further improve application of these data towards bioprocess development.

Evaluation of guanidine-based multimodal anion exchangers for protein selectivity and orthogonality

In this paper, we examined the chromatographic behavior of a new class of guanidine-based multimodal anion exchange resins. The selectivities and protein recoveries on these resins were first evaluated using linear gradient chromatography with a model acidic protein library at pH 5, 6 and 7. While a single-guanidine based resin exhibited significant recovery issues at high ligand density, a bis-guanidine based resin showed high recoveries of all but two of the proteins evaluated in the study. In addition, the bis-guanidine resin showed a more pH dependent selectivity pattern as compared to the low density single-guanidine resin. The salt elution range for the low density single-guanidine and bis-guanidine resins was also observed to vary from 0.250 to 0.621 M and 0.162 to 0.828 M NaCl, respectively. A QSAR model was then developed to predict the elution behavior of these proteins on the guanidine prototypes at multiple pH with overall training and test scores of 0.88 and 0.85, respectively. In addition, molecular dynamics simulations were performed with these ligands immobilized on a self-assembled monolayer (SAM) to characterize their conformational preferences and to gain insight into the molecular basis of their chromatographic behavior. Finally, a recently developed framework was employed to evaluate the separability of the bis-guanidine resin as well as its orthogonality to the multimodal cation exchanger, Nuvia cPrime. This evaluation was carried out using a second model protein library which included both acidic and basic proteins. The results of this analysis indicated that the bis-guanidine prototype exhibited both higher pair separability (0.73) and pair enhancement (0.42) as compared to the less hydrophobic commercial Nuvia aPrime 4A with pair separability and enhancement factors of 0.57 and 0.22, respectively. The enhanced selectivity and orthogonality of this new multimodal anion exchange ligand may offer potential opportunities for bioprocessing applications.

A Screening Method for 30 Drugs in Hair Using Ultrahigh-Performance Liquid Chromatography Time-of-Flight Mass Spectrometry

The objectives of this study were to develop and to validate a qualitative screening method that met the new Society of Hair Testing (SoHT) guideline criteria for thresholds. Extraction of 20 mg hair was performed by a previously validated procedure using overnight incubation in a mixture of methanol:acetonitrile:formiate buffer pH 3 (10:10:80). Analysis was performed on an Agilent 6540 quadrupole time-of-flight mass spectrometer in combination with an Agilent 1290 Infinity ultrahigh-performance liquid chromatography system. Separation was achieved with a 12-minute linear gradient chromatography on a high-strength silica T3 column at acidic conditions. An in-house database containing 30 compounds from the groups amphetamines, opiates, opioids, cocaine, benzodiazepines, and other sedatives including 6 deuterated internal standards was built by analyzing solutions from certified standards. Data were extracted using mass accuracy of ± 10 ppm, retention time deviation of ± 0.15 minutes, and area of ≥ 30,000 counts. Identification was based on scoring of retention time, accurate mass measurement, and isotopic pattern. Validation included selectivity, repeatability of analyte area, and the scoring parameters at the proposed thresholds and a method comparison with the present liquid chromatography-mass spectrometry-mass spectrometry method using 50 authentic hair samples. A daily cutoff calibrator was used to identify positive samples. All cutoffs could be met with imprecisions of less than 5% for most parameters and analytes. Hair from drug-free subjects did not produce any positive results and the method comparison agreed in more than 90% of the cases. We conclude that the developed method meets the criteria of the new SoHT guidelines for screening cutoffs. Even though no thresholds have been suggested for benzodiazepines, we conclude that thresholds between 0.05 and 0.1 ng/mg should be sufficient to determine regular use of these substances.

Selective displacement chromatography in multimodal cation exchange systems

A library of displacer analogues with varying degrees of electrostatic, hydrophobic and hydrogen bonding moieties was evaluated for their ability to enhance the selectivity of multimodal (MM) chromatography under high loading conditions. The library was screened for displacement of model proteins using a robotic liquid handling system and selective batch separations were achieved for proteins that were inseparable with linear gradient chromatography. Trends in protein displacement were identified and displacers with higher hydrophobicity and net charge exhibited improved protein displacements. Proteins that interacted with the resins primarily via electrostatic interactions were more readily displaced than those that possessed a significant hydrophobic contribution to their binding. In addition, multimodal displacers were found to be more selective than single mode electrostatic displacers. Column chromatography studies were also carried out and baseline separations were achieved for model protein pairs using selective displacement. Finally, operation of these columns in the desorption mode resulted in baseline separation of model proteins which were not separable by selective displacement chromatography. This study indicates that the inherent selectivity of MM resins can be augmented by the selectivity of the displacer under non-linear competitive binding conditions, creating new opportunities for protein separations not possible using traditional gradient operations.

The thermodynamic limit of linear gradient chromatography

The thermodynamic limit of the elution profile for solutes in linear gradient chromatography is obtained from the analytical solution of the equation for the ideal model of chromatography, Eq. (12). This limit is of great interest in both preparative and analytical chromatographies because it specifies the maximum possible concentration profile that can be achieved at elution. Elution profiles that are obtained from simulated experiments of the equilibrium dispersive model, Eq. (8), are compared with predictions made by the presented theory as well as the theory by Poppe [11]. It is found that for short injection times the simulated experimental peak is Gaussian like and its width agrees very well with the theory of Poppe. When the injection time increases, the experimental elution profile gradually approaches the profile that is obtained as the thermodynamic limit.

Investigation of protein binding affinity in multimodal chromatographic systems using a homologous protein library

A library of cold shock protein B mutant variants was employed to examine differences in protein binding behavior in ion exchange and multimodal chromatography. Single site mutations introduced at charged amino acids on the protein surface resulted in a homologous protein set with varying charge density and distribution. The retention times of the mutants varied significantly during linear gradient chromatography in both systems. The majority of the proteins were more strongly retained on the multimodal cation exchange resin as compared to the traditional cation exchanger. Further, the elution order of the mutants on the multimodal resin was different from that obtained with the ion exchanger. Quantitative structure–property relationship models generated using a support vector regression technique were shown to provide good predictions for the retention times of protein mutants on the multimodal resin. A coarse-grained ligand docking package was employed to examine the various interactions between the proteins and ligands in free solution. The multimodal ligand was shown to utilize multiple interaction types to achieve stronger retention on the protein surface. The use of this protein library in concert with the qualitative and quantitative analyses presented in this paper provides an improved understanding of protein behavior in multimodal chromatographic systems.

Investigation of protein binding affinity and preferred orientations in ion exchange systems using a homologous protein library

A library of cold shock protein B (CspB) mutant variants was employed to study protein binding affinity and preferred orientations in cation exchange chromatography. Single site mutations introduced at charged amino acids on the protein surface resulted in a homologous protein set with varying charge density and distribution. The retention times of the mutants varied significantly during linear gradient chromatography. While the expected trends were observed with increasing or decreasing positive charge on the protein surface, the degree of change was a strong function of the location and microenvironment of the mutated amino acid. Quantitative structure-property relationship (QSPR) models were generated using a support vector regression technique that was able to give good predictions of the retention times of the various mutants. Molecular descriptors selected during model generation were used to elucidate the factors affecting protein retention. Electrostatic potential maps were also employed to provide insight into the effects of protein surface topography, charge density and charge distribution on protein binding affinity and possible preferred binding orientations. The use of this protein mutant library in concert with the qualitative and quantitative analyses presented in the article provides an improved understanding of protein behavior in ion exchange systems.

Investigation of protein retention and selectivity in HIC systems using quantitative structure retention relationship models

In the present work, the effect of stationary phase resin chemistry and protein physicochemical properties on protein binding affinity in hydrophobic interaction chromatography (HIC) was investigated using linear gradient chromatography and quantitative structure-retention relationship (QSRR) modeling. Linear gradient experiments were carried out for a set of model proteins on four different HIC resins having different backbone and ligand chemistry. The retention data exhibited significant differences in protein binding affinity, not only across the phenyl and butyl ligand chemistries, but also for the different backbone chemistries found in the Sepharose (cross-linked agarose) and the Toyopearl 650 M (polymethacrylate) series of resins. QSRR models based on a Support Vector Machine (SVM) approach were developed for the linear retention data using molecular descriptors based on protein crystal structure and primary sequence information as well as a set of new hydrophobicity descriptors based on the solvent accessible protein surface area. The results indicate that the QSRR models were successfully able to capture and selectivity predict the changes observed in these systems. Furthermore, the new descriptors resulted in physically interpretable models of protein retention and provided insights into the factors influencing protein affinity in these different HIC systems. The approach put forth in this study provides a framework for developing predictive tools and for gaining insight into protein selectivity in hydrophobic interaction chromatography.

Nonlinear Multicomponent Gradient Chromatography in Metal Affinity Systems

ABSTRACT In this paper the metal affinity interaction chromatography (MAIC) model is employed in concert with appropriate mass transport equations to study preparative linear gradient chromatography in immobilized metal affinity chromatography (IMAC) systems. The MAIC model accounts for the nonlinear adsorption of proteins and mobile phase modulators (e.g., imidazole), and is shown to accurately predict gradient separations of proteins under overloaded conditions. Experimental and simulation results indicate that the concentration-dependent sorption of imidazole and protein-imidazole interference effects can severely deform linear gradients in IMAC systems. The steric accessibility and displacer characteristics of imidazole together with multicomponent interference effects can lead to unusual protein elution profiles and the spiking of imidazole between the feed components. Due to their ability to act as displacers, these imidazole spikes can sharpen protein tails, decreasing the interface shock layer thickness and improving resolution. Finally, iterative optimization schemes are employed to study the influence of the transient displacer characteristics of imidazole shocks on the optimum operating conditions.

Overload Quasi-Static Linear Gradient Chromatography: Theory Versus Hydroxyapatite High-Performance Liquid Chromatography

Abstract The earlier theory of quasi-static linear gradient chromatography under overload conditions is improved. The quasi-crystalline phase model for the molecules adsorbed on the adsorbent surfaces in the column is specified; this model was introduced earlier, and it is used in the present theory. A close experimental verification of the theory is carried out for the case of hydroxyapatite high-performance liquid chromatography using both turkey and chicken egg white lysozymes as probes. It can be deduced that a quasi-crystalline phase of lysozyme molecules, in fact, is realized on the hydroxyapatite surfaces in the column. The mutual interaction energy per molecule of lysozyme occurring provided the maximum possible contact with other molecules is made on the hydroxyapatite surface can be estimated to be larger than 0.2–0.4 kcal/mol.

A Fundamental Structure of the General Theory of Overload Quasi-Static Linear Gradient Chromatography

Abstract The earlier theory of overload linear gradient chromatography developed over 15 years ago by using an ideal molecular model with infinite dimensions is rearranged and improved in order for it to be compatible with the new concepts that are involved in the general theory of gradient chromatography developed recently. By applying the ideal molecular model, the structure of the overload chromatographic theory is considerably reduced, emphasizing the fundamental structure. Chromatograms with a shape close to a right-angled triangle with tailing are often experienced under overload condition. On the basis of the present theory, this type of chromatogram can be explained in terms of the occurrence of a quasi-crystalline phase of the adsorbed molecules that are repulsively interacting with one another on the adsorbent surfaces in the column.

A Specification of the General Theory of Quasi-Static Linear Gradient Chromatography: Relationship with the Overload Chromatography Theory in the Presence of Mutual Molecular Interactions

Abstract The earlier theory of quasi-static linear gradient chromatography with small sample loads is partially specified. On the basis of this study, the relationship between the theory of small load gradient chromatography and the approximate theory of overload gradient chromatography which was developed several years ago is also specified; this will promote a better understanding of the overload theory.

Further Investigations on the General Theory of Quasi-Static Linear Gradient Chromatography

Abstract The earlier theory of quasi-static linear gradient chromatography is still generalized. The present theory is valid whatever may be the initial location of the sample molecules on the column, and the condition imposed upon the quasistatic chromatography is less severe in this theory than earlier. The earlier theory represents a special case of the present theory. The universal continuity equation for any quasi-static chromatography is. derived, from which the continuity equation that causally describes the chromatographic process can be derived for both gradient and isocratic (or stepwise) chromatographies.

General Theory off Quasi-Static Linear Gradient Chromatography

Abstract The earlier theory of hydroxyapatite chromatography is extended to a general theory of quasi-static linear gradient chromatography. The present theory is partially valid for the nonquasi-static case.

Characterization of chromatin-bound erythrocyte histone V (f2c). Synthesis, acetylation, and phosphorylation

Synthesis and enzymatic modification of histone V was 1 order of magnitude lower in mature gander erythrocytes as compared with immature enriched cells hwich were capable of DNA synthesis. Application of shallow, linear gradient chromatography was used to demonstrate qualitative changes as well. This technique permitted the separation of newly synthesized and phosphorylated histone V from older, less phosphorylated molecules but did not discriminate between acetylated species. The most easily eluted fractions were those most recently synthesized, acetylated, and phosphorylated. While lysine chased into the other subfractions of histone V, phosphate did not, indicating a dephosphorylation step in the immature cells. Acetylation of histone V which occurs at a very low level was closely related to its synthesis. No differences in molecular weights or amino acid compositions were apparent, and behavior on polyacrylamide gels was similar to whole histone V. It is proposed that phosphorylation of histone V may play an important role in the modulation of the effect of histone V in immature cells on condensation and template restriction of chromatin which occurs in the terminal stages of differentiation of the avian erythroid cells.

Microheterogeneity of cross-linked γ dimers isolated from bovine stabilized fibrin

Bovine stabilized fibrin was reduced, carboxymethylated and separated by chromatography on a Sepharose 4B column. The fraction containing cross-linked γ dimers was then subjected to linear gradient chromatography on a CM-52 column. On this column, the γ dimers were separated into an adsorbed and unadsorbed fraction. The components in these fractions were designated as the γ-1 and γ-2 dimers. They each gave a single band on SDS-polyacrylamide gel electrophoresis and both had a molecular weight of 90,000 (±2,000). The identities of the γ-1 and γ-2 dimers were also shown by their amino acid compositions, terminal residues and tryptic and plasmic maps. However, they differed in electrophoretic mobilities on gels at pH 8.3 and pH 3.6 and in carbohydrate composition. The γ-1 dimer was slightly acidic and contained more hexoses and glucosamine than the γ-2 dimer. These results indicate that the characteristics of the bovine monomeric γ chains, γ-1 and γ-2, previously reported by Gerbeck et al ., are transferred to their corresponding cross-linked γ dimers, formed in the stabilization of fibrin.

Isolation of porcine proinsulin from crystalline porcine insulin.

After recent discoveries of bovine, porcine, rat and human proinsulin, extensive efforts have been made for their physiological and biological significance. Only a few informations are available concerning them at present, because of shorter quantity of the hormones.An isolation method of porcine proinsulin from crystalline porcine insulin with easy performance has been described.Repeated gel chromatographic separations of porcine insulin on Bio-Gel column yielded crude porcine proinsulin and its intermediate form. A single band component with corresponding electrophoretic mobility of porcine proinsulin was obtained by linear gradient chromatography of this crude material on DEAE-cellulose column, and was converted to insulin by trypsin digestion. All of DEAE-cellulose chromatographic fractions having more basic components than insulin could be converted to insulin by the trypsin treatment. Amino acids analysis of this single band component appeared to be the same amino acids composition of known porcine proinsulin.