Altering the mobile phase composition to enhance self-disproportionation of enantiomers in achiral chromatography.

The influence of mobile phase composition on the efficiency of enantiomer separation by achiral chromatography (ACh) was investigated. The separation was induced by the phenomenon of self-disproportionation of enantiomers (SDE) triggered by their homo and hetero-chiral interactions in an achiral environment. Typically, SDE occurs in apolar mobile phases of weak elution strength, which causes the separation time to extend and the process productivity to deteriorate. To mitigate that effect, we altered the content of a strong solvent (modifier) in the mobile phase by use of a solvent gradient in which the target enantiomer was separated in the presence of the weak solvent, whereas the unresolved mixture of enantiomers was eluted by increasing the modifier content in the mobile phase. This enabled accelerating the solute elution while preserving the separation selectivity. The approach was examined for the separation of nonracemic mixtures of two structurally different compounds that exhibited the SDE effect in ACh, i.e., metalaxyl (MX) and methyl p-tolyl sulfoxide (MTSO). The target compound of the separation was the more abundant enantiomer in the enantiomeric mixture. The process realization was preceded by the determination of the effect of the modifier content on the separation yield for enantiomeric mixtures of MX and MTSO of different enantiomeric excess (ee). In the case of MX, yield of the pure target enantiomer varied from 2 %, for the maximum concentration of the modifier, to 45 % for the minimum modifier concentration and the largest ee used in the experiments. In the case of MTSO, the yield varied from minimum 40 % to maximum 66 %. To predict the process, we employed a dynamic model, in which underlying thermodynamic dependencies were implemented.

Flow behavior of protein solutions in a lab-scale chromatographic system.

A fluid dynamics model has been developed to describe flow behavior in a lab-scale chromatographic system dedicated for protein processing. The case study included a detailed analysis of elution pattern of a protein, which was a monoclonal antibody, glycerol, and their mixtures in aqueous solutions. Glycerol solutions mimicked viscous environment of the concentrated protein solutions. The model accounted for concentration dependences of solution viscosity and density, and dispersion anisotropy in the packed bed. It was implemented into a commercial computational fluid dynamics software using user-defined functions. The prediction efficiency was successfully verified by comparing the model simulations in the form of the concentration profiles and their variances with the corresponding experimental data. The contribution of the individual elements of the chromatographic system to protein band broadening was evaluated for different configurations: for the extra-column volumes in the absence of the chromatographic column, for the zero-length column without the packed bed and for the column containing the packed bed. The influence of the operating variables, including: the mobile phase flowrate, the type of the injection system, i.e., the injection loop capillary or the superloop, the injection volume and the length of the packed bed, on band broadening of the protein was determined under nonadsorbing conditions. For protein solutions having viscosity comparable with the mobile phase, the flow behavior either in the column hardware or in the injection system made major contributions to band broadening, which depended on the type of the injection system. For highly viscous protein solution, the flow behavior in the packed bed exerted a dominant influence on band broadening.

Coupling of chromatography and precipitation for adjusting acidic variant content in a monoclonal antibody pool.

The acidic charge variants (av) of monoclonal antibodies (mAb) are often reported to have reduced therapeutic potency compared with the main (mv) and basic variants (bv), therefore reduction in the av content in mAb pools is often prioritized over reduction in the bv content. In previous studies we described two different methods for reducing the av content, which were based on either ion exchange chromatography or selective precipitation in polyethylene glycol (PEG) solutions. In this study, we have developed a coupled process, in which advantages of simplicity and ease in realization of PEG-aided precipitation and high separation selectivity of anion exchange chromatography (AEX) were exploited. The design of AEX was supported by the kinetic-dispersive model, which was supplemented with the colloidal particle adsorption isotherm, whereas the precipitation process and its coupling with AEX was quantified by simple mass balance equations and underlying thermodynamic dependencies. The model was used to assess the performance of the coupling of AEX and precipitation under different operating conditions. The advantage of the coupled process over the stand-alone AEX depended on the demand for the av reduction as well as the initial variant composition of the mAb pool, e.g., the improvement in the throughput provided by the optimized sequence of AEX and PREC varied from 70 to 600% for the initial av content changed from 35 to 50% w/w, and the reduction demand changed from 30 to 60%.

Protein association on multimodal chromatography media.

The phenomenon of protein-protein association on multimodal chromatography resins was described for two different case study examples. The adsorption pattern of single-component solutions of calcium-rich alpha-lactalbumin (aLaCa) and calcium-depleted alpha-lactalbumin (aLa) and their mixtures with bovine serum albumin was determined on a multimodal anion-exchange chromatography medium. In single-component solutions, both aLaCa and aLa exhibited identical adsorption behavior at low resin loadings, whereas at high loadings the adsorption strength of aLa markedly exceeded that of alaCa. In binary mixtures, the adsorption of BSA enhanced at high concentrations of aLa or aLaCa in the adsorbed phase. The unusual adsorption patterns observed were attributed to the tendency of the proteins for molecular association in the adsorbed phase in single and binary solutions. The phenomena was examined for different pH of the solution: pH 6, 7, 8, and different solvent environments: phosphate buffer (PB), bis tris buffer (BT), 100 mM NaCl in BT and bis tris propane buffer (BTP). The strongest effect was observed for PB and for 100 mM NaCl in BT. Its occurrence was also evidenced for other case study example, i.e., adsorption of single-component solutions and binary mixtures of a monoclonal antibody (mAb) and lysozyme (LYZ) on a multimodal cation-exchange chromatography medium. The enhancement of adsorption of mAb was observed at high concentrations of LYZ in the adsorbed phase. To quantify the underlying effects, a mechanistic model was used, which accounted for both protein association and exclusion resulting from attractive and repulsive protein-protein iterations in the adsorbed phase.

Separation of non-racemic mixtures of enantiomers by achiral chromatography.

The phenomenon of partial separation of enantiomeric mixtures in achiral chromatography (ACh) has already been documented for a wide variety of chiral compounds. It is attributed to the so-called effect of self-disproportionation of enantiomers (SDE). However, quantitative description of the SDE mechanism underlying adsorption of enantiomers on achiral surfaces is still incomplete, which hinders the application of that technique for large-scale separations. In this study, a mechanistic model for description of retention behavior of SDE-phoric compounds in silica-based ACh has been developed along with a procedure for fast determination of the model parameters. The model assumes formation of associates of chiral molecules, which occurs due to homo and hetero-chiral interactions in the adsorbed phase. The ability of the model to reproduce band profiles was verified for enantiomeric mixtures of three structurally different chiral compounds.

Preferential precipitation of acidic variants from monoclonal antibody pools.

Microheterogeneity of monoclonal antibodies (mAbs) can impact their activity and stability. Formation of charge variants is considered as the most important source of the microheterogeneity. In particular, controlling the content of the acidic species is often of major importance for the production process and regulatory approval of therapeutic proteins. In this study, the preferential precipitation process was developed for reducing the content of acidic variants in mAb downstream pools. The process design was preceded by the determination of phase behavior of mAb variants in the presence of different precipitants. It was shown that the presence of polyethylene glycol (PEG) in protein solutions favored precipitation of acidic variants of mAbs. Precipitation yield was influenced by the variant composition in the mAb feed solutions, the concentration of the precipitant and the protein, and the ionic strength of the solutions. To improve yield, multistage precipitation was employed, where the precipitate was recycled to the precipitation process. The final product was a mixture of supernatants pooled together from the recycling steps. Such an approach can be potentially used either instead or in a combination with chromatography for adjusting the acidic variant content of mAbs, which can benefit in improvement in throughput and reduction in manufacturing costs.

Dissociation events during processing of monoclonal antibodies on strong cation exchange resins.

The phenomenon of pH excursion was demonstrated for pH gradient elution of monoclonal antibodies (mAbs) on strong cation exchange resins under high overloading conditions. The mAbs differed in molecular structure and isoelectric point, and the resins in matrix structure and ligand density. In all cases, elution of the proteins was accompanied with distortion of the concentration, pH and conductometric profiles. To elucidate that phenomenon, titration curves were generated for liquid solutions of the proteins as well as for suspensions of the resins with the proteins adsorbed on their surface. The course of the curves was found to be affected by the presence of the proteins both in liquid and adsorbed phases. The effect enhanced with increase in the initial pH of the binding buffer and in the protein concentration. To quantify that phenomenon, a mechanistic model was used, which accounted for the protein dissociation in both phases. The model reproduced the titration curves and the observed trends in changes of their courses. The simulation results indicated that the pattern of pH transitions recorded for different mAbs on the resins mostly depended on their adsorption affinity.

Separation of charge variants of a monoclonal antibody by overloaded ion exchange chromatography.

A procedure for adjusting the content of charge variants of monoclonal antibody by ion exchange chromatography has been developed. The band splitting phenomenon was utilized to split the protein load into two parts, i.e., the flowthrough and bound fractions, which were either enriched or depleted with some of variants. The phenomenon was triggered by thermodynamic effects resulting from oversaturation of the resin binding sites at high column loadings as well as from kinetic effects arising from limited rates of mass transport. Cation exchange chromatography (CEX) and anion exchange chromatography (AEX) separations were examined, with the reverse order of the variant elution: acidic, main, basic in CEX, and basic, main, acidic in AEX, and the corresponding reverse enrichment tendency in the collected fractions. The separations were performed by pH gradient, whose course was simplified to two stages: isocratic loading and washing at mild pH to load and partly elute the protein, followed by a rapid pH change towards non-binding conditions to desorb the remains of the protein load. To improve yield of the operation, possibility of recycling of waste fractions was considered. To predict the process performance, a dynamic model was developed, which accounted for both adsorption kinetics and thermodynamics.

Influence of the geometry of extra column volumes on band broadening in a chromatographic system. Predictions by computational fluid dynamics.

A computational fluid dynamics method was used for prediction of flow behavior and band profiles of small- and macro-molecule compounds eluting in extra-column volumes (ECV) of an Äkta chromatographic system. The model compounds were: acetone, bovine serum albumin and an antibody. The construction of ECV was approximated by different types of geometries, starting from the simplest two-dimensional (2D) arrangement consisting of a straight capillary tube, and ending with a three-dimensional system (3D), which accounted for the flow path curvature of individual elements of ECV, including: injection loop capillary, multi-way valve, connecting capillary and detector cell. The accuracy of the model predictions depended on the flow path length and the eluent flowrate. 2D-geometry models reproduced pretty well the shapes of band profiles recorded at the lowest eluent flowrate used, but they failed for increased flowrates. The 3D-geometry model was found to be sufficiently accurate for all conditions investigated. It was exploited to analyze band broadening in the individual ECV elements. The simulation results revealed that the flow behavior in the injection loop capillaries strongly influenced the shape of band profiles, particularly at higher eluent velocities. This was attributed to the formation of Dean vertices triggered by centrifugal forces in curved parts of the eluent flow path.

A case study of the mechanism of unfolding and aggregation of a monoclonal antibody in ion exchange chromatography.

A mechanistic model for describing unfolding of a monoclonal antibody (mAb) in ion exchange chromatography has been developed. The model reproduced retention behavior characteristic for conformational changes of antibodies upon adsorption, including: multi-peak elution, aggregate formation, and recovery reduction. Two competitive paths in the adsorption mechanism of the unfolded protein were assumed: refolding in the adsorbed phase to the native form followed by its desorption, or direct desorption followed by instantaneous aggregation in the liquid phase. The reduction in recovery of the eluted protein was attributed to spreading of the unfolded protein on the adsorbent surface, which enhanced the binding affinity. The model was formulated based on the analysis of retention behavior of a model mAb that was eluted in pH gradients on a strong cation exchange resin. The pH profile was found to be distorted in the presence of the protein, which was ascribed to dissociation of ionizable groups of the protein in the adsorbed phase. Since the protein retention was strongly pH dependent, that phenomenon was also accounted for in mathematical modeling. A series of independent experiments was designed to evaluate the model parameters that quantified the process thermodynamics and kinetics: the Henry constants of the native, unfolded, spread and aggregated forms of the protein along with underlying kinetic coefficients. The model was efficient in reproducing the retention pattern of the protein and the aggregate content in eluting band profiles. After proper calibration, the model can potentially be used to quantify protein unfolding and elution in other ion exchange systems.

A high-throughput method for fast detecting unfolding of monoclonal antibodies on cation exchange resins.

A fast method for assessing the stability of monoclonal antibodies (mAbs) adsorbed on ion exchange resins has been developed. The method exploited a real time polymerase chain reaction equipment to determine the temperature of protein phase transition, i.e., the so called melting temperature, based on differential scanning fluorimetry. Changes to the melting temperature were screened under various adsorption conditions and correlated with the protein stability upon adsorption. The method was tested for two different mAbs bound to various types of strong cation exchangers at different pH and loading concentrations. The mAbs destabilized upon adsorption due to strong binding, which manifested itself in aggregate formation and recovery reduction. The phenomenon depended on the resin type and binding conditions. However, regardless of the process conditions and resins used, drop in the melting temperatures to a critical value of about 30° could serve as an indicator of destructive changes in the protein structure in the adsorbed phase. The measurements were simultaneously accomplished for a number of samples with very small material consumption. Therefore, the method may be applied for screening resins and operating variables for a given mAb to exclude conditions that induce structure destabilization and aggregation.

Effects of negative and positive cooperative adsorption of proteins on hydrophobic interaction chromatography media.

The adsorption behavior of the model proteins: alpha-Lactalbumin, Bovine Serum Albumin, Lysozyme, and a monoclonal antibody, in single component and in binary mixtures, was investigated on two different hydrophobic interaction chromatography resins using both static and dynamic methods. A kinetic model of the adsorption process was developed, which accounted for protein unfolding and intermolecular interactions in the adsorbed phase. The latter incorporated positive cooperative interactions, resulting from preferred and multilayer adsorption on the adsorbent surface, as well as negative cooperative interactions attributed to exclusion effects due to size exclusion and repulsion. Cooperative adsorption resulted in negative or positive deviations from the Langmuir model for both single and multicomponent isotherms. The model was used to assess possible contributions of different adsorption mechanisms of proteins and their structurally different forms to the overall adsorption pattern, as well as to simulate chromatographic band profiles under different loading conditions. For proteins with unstable structure, the overall adsorption isotherm was dominated by binding of unfolded species at low surface coverage and by positive cooperative adsorption at high surface coverage. Furthermore, regardless of structural stability, exclusion effects influenced strongly adsorption equilibrium, particularly at low surface coverages. In case of chromatographic elution, i.e. under dynamic conditions, unfolding, negative cooperative adsorption, and kinetic effects governed the retention behavior and determined peak shapes, whereas the effect of positive cooperative adsorption was negligible.

Mechanism of nutrition activity of a microgranule fertilizer fortified with proteins.

BACKGROUND: A microgranule fertilizer was designed for localized fertilization of soil with controlled release of nutrients. The microgranule matrix was fortified with proteins, which were obtained from food industry byproducts or waste, i.e., whey protein from milk serum, soy protein from soy isolate and egg white protein from chicken egg white powder. The mechanism of the protein decomposition and migration of micro and macromolecule compounds through two different model soil systems was investigated. The potential of the protein fortified fertilizer for localized fertilization of the potted maize seeds was evaluated. RESULTS: The study revealed that proteins slowly diffused through soil with simultaneous degradation, which was accompanied with release of ammonia ions. The highest concentration of proteins and degradation products was found in a close vicinity of the microgranule. The microgranules were used as a local fertilizer for maize seeds in the pot experiments. The experiments confirmed statistically significant improvement in root density of maize plant compared to control group. CONCLUSIONS: Byproducts or waste of food industry, such as the milk serum and soy can be used as a source of proteins that degrade in soil without a pretreatment. The degradation is accompanied with formation of ammonium ions, which can be utilized by plants as a nitrogen source. The fertilizer microgranule should be placed in a close vicinity to the plant seed, since the maximum of the protein concentration and ammonia ions is reached at a very close distance from the microgranule.

Scale up of a chromatographic capture step for a clarified bacterial homogenate - Influence of mass transport limitation and competitive adsorption of impurities.

A model-based approach for scaling up chromatographic capture step was developed. The purification of human basic fibroblast growth factor protein 2 (FGF2) from an E. coli homogenate on a cation exchange resin was selected as a case study. Non-ideal effects accompanying the capture operation were examined, including: reduction in the protein diffusivity in the presence of the homogenate, competitive adsorption between FGF2 and undefined impurities, and flow behavior in external column volumes. The viscosity of the homogenate was measured as a function of dilution degree and shear stress, and its contribution to the diffusivity reduction was quantified. A dynamic model was formulated which accounted for underlying kinetic and thermodynamic dependencies. The model parameters were determined for a lab scale system using a small 2-mL column. The model was successfully used to scale up the capture operation from the lab scale column to a preparative bench scale column of about 1 L volume.

Effect of flow behavior in extra-column volumes on the retention pattern of proteins in a small column.

Experimental and theoretical analysis of deformation of band profiles in extra-column volumes (ECV) was performed, and its influence on the retention pattern of proteins in a small chromatographic column was quantified. Several macromolecule and small-molecule compounds, and their mixtures were eluted from a chromatographic system in the absence and presence of the column. The peak deformation in ECV was attributed to non-uniform velocity distribution in the radial direction in connecting capillaries. The phenomenon enhanced with increasing molecular weight of the model compound, when radial diffusion dominated the mechanism of band spreading. The band shape was also affected by the geometry of the injection system used, i.e., an injection loop capillary or a superloop. The phenomenon vanished for a small molecule compound, for which plug flow conditions could be established. The difference in flow behaviour of the macromolecule and small-molecule compounds caused them to migrate with different velocities in ECV, which resulted in partial separation of their bands. The ECV effect influenced the retention behaviour of macromolecules in a small column; it caused tailing of peaks and asymmetry of breakthrough curves. To describe the elution profiles in ECV and in the column, a mathematical model was used which accounted for non-ideality of the flow pattern. The model reproduced accurately band profiles of macromolecules within a range of relatively low velocities, typical however for protein chromatography.

Retention Behavior of Polyethylene Glycol and Its Influence on Protein Elution on Hydrophobic Interaction Chromatography Media.

The retention behavior of polyethylene glycol (PEG) on different types of hydrophobic interaction chromatography (HIC) resins containing butyl, octyl, and phenyl ligands was analyzed. An incomplete elution or splitting of the polymer peak into two parts was observed, where the first one was eluted at the dead time of the column, whereas the second one was strongly retained. The phenomenon was attributed to conformation changes of the polymer upon its adsorption on hydrophobic surface. The effect enhanced with increasing molecular weight of the polymer and hydrophobicity of the HIC media. Addition of PEG to the mobile phase reduced binding of proteins to HIC resins, which was demonstrated with two model systems: lysozyme (LYZ) and immunoglobulin G (IgG), and their mixtures. In case of LYZ, the presence of PEG caused reduction in the protein retention, whereas for IgG-a decrease in efficiency of the protein capture. The effect depended on the adsorption pattern of PEG; it was pronounced in the systems in which conformational changes of the polymer were suggested to occur.

Prediction tool for loading, isocratic elution, gradient elution and scaling up of ion exchange chromatography of proteins.

An efficient mathematical tool for the design and scaling up of protein chromatography is suggested, in which the model parameters can be determined quickly over a wide operating space without large material investments. The design method is based on mathematical modelling of column dynamics and moment analysis. The accuracy of the dynamic models that are most frequently used for simulations of chromatographic processes is analyzed, and possible errors that can be generated using the moment analysis are indicated. The so-called transport dispersive model was eventually employed for the process simulations. The model was modified to account for the protein dispersion in void volumes of chromatographic systems. The manner of the model calibration was suggested, which was based on a few chromatographic runs and verified over a wide space of the operating parameters, including composition and flow rate of the mobile phase, column dimensions, residence time, and mass loading. The model system for the study was ion-exchange chromatography. The analysis was performed based on the elution profiles of basic fibroblast growth factor 2 and lysozyme, on two different IEX media.

Effect of mass overloading on binding and elution of unstable proteins in hydrophobic interaction chromatography.

Adsorption behavior of unstable proteins, i.e., bovine serum albumin and α-lactalbumin, has been studied on a hydrophobic interaction chromatography medium under mass overloading conditions at different kosmotropic salt concentrations in the mobile phase. A mechanistic model has been formulated and used to describe kinetics and thermodynamics of protein interactions with the adsorbent surface. The model assumed two-site binding adsorption and reversible protein unfolding, which allowed predicting the inhibition of protein unfolding at high column loadings. A simplified procedure for the determination of model parameters has been developed, which was based on the inverse method. The model was successfully used to reproduce the pattern of chromatographic elution as well as the course of breakthrough curves. The model formulation was supported by Nano Differential Scanning Fluorimetry measurements, which were exploited to determine the protein stability in the liquid and adsorbed phases at different column loadings and salt concentrations.

A shortcut method for evaluation of protein deposition onto the membrane surface in crossflow ultrafiltration.

In this study, a procedure for quantifying the surface deposition of proteins in crossflow ultrafiltration has been developed. The procedure consists of determining the protein adsorption behavior onto the membrane surface from a few dynamic measurements performed in a nonfiltration and a filtration mode, and evaluating the concentration polarization (CP) layer thickness based on the adsorption data. To predict the interdependence between the protein adsorption and CP, a simplified mathematical model has been formulated. The model was used to assess the protein adsorption and thus yield reduction in the ultrafiltration process at different protein concentration in the solution. As a case study, ultrafiltration of aqueous solutions of BSA and lysozyme (LYZ) was examined on a polyethersulfone membrane with the molecular weight cutoff of 10 or 100 kDa. The protein concentration in the solutions varied within a relatively low concentration range, i.e. below 10 mg mL-1, characteristic for solvent exchange between sequential operations of protein purification by chromatography and extraction. Both proteins markedly differed in the mechanism of surface deposition; for BSA hydrophobic interactions were suggested to be dominant, whereas in case of LYZ electrostatic interactions contributed the most to the deposition mechanism. The effect of additives of the protein solutions, i.e. inorganic salts, PEG, and urea depended on the adsorption mechanism and was also specific for each protein. Nevertheless, the proposed procedure performed well in the evaluation of surface deposition and yield reduction, regardless of the protein type and its solvent environment.

Protein separation in carousel multicolumn setup. Performance analysis and experimental validation.

To overcome limitations of periodic separations of proteins in batch chromatographic columns Carousel Multi-Column Setup (CMS) has been recently suggested and theoretically analyzed in a previous study (R. Bochenek, W. Marek, W. Piatkowski, D. Antos, J. Chromatogr. A, 1301 (2013) 60-72). In this system, feed and mobile phase streams are subsequently delivered through parallel columns to mimic their countercurrent movement with respect to the fluid flow. All fluxes in the system are synchronized to ensure continuous feed delivery, which however causes reduction in the size of the operating window compared to batchwise-operating systems. In this study to improve the performance of CMS, additional process variables have been considered, such as the flow rate gradient and feed concentration. Though altering both variables allowed improving the separation selectivity and extending the operating window, the feed concentration appeared to be the most influential parameter affecting the process performance. Moreover, a procedure for practical realization of protein separations in CMS has been developed, including hints about the process design, configuration of columns and detectors, and use of pumps. As the case study, the separation of a ternary mixture of proteins, i.e., cytochrome C, lysozyme and immunoglobulin G, on hydrophobic interaction columns was used. A target product was a protein with intermediate adsorption strength that was isolated out of a more and less strongly adsorbed compound.