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.

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.

Overcoming solubility limits in overloaded gradient hydrophobic interaction chromatography.

The impact of the solubility limits on the performance of gradient protein chromatography has been studied. As a case study elution of model protein, i.e., lysozyme, on hydrophobic interaction media has been selected. A dependence of the protein solubility and crystallization kinetics on the content of cosmotropic salt in the mobile phase has been determined. Moreover, adsorption properties of the protein versus the mobile phase composition have been quantified. A model of chromatographic column dynamics has been developed which incorporated the mass transport kinetics accompanying both adsorption and crystallization processes. The model was used to study the influence of operating parameters such as flowrate and concentration loading on the solubility pattern inside the column and the separation performance. The analysis performed indicated existence of supersaturation regions for which, due to slow kinetics of crystallization, chromatographic process could be performed under conditions of strong concentration overloading while avoiding undesirable effects of flow blockage in chromatographic systems.

Adsorption behavior of proteins on temperature-responsive resins.

The adsorption behavior of proteins on thermo-responsible resins based on poly(N-isopropylacrylamide) and its copolymer containing an anionic co-monomer has been investigated. The influence of the polymer composition, i.e., the content of the co-monomer and crosslinker on the thermo-sensitivity of the protein adsorption has been quantified. The properties of ungrafted polymer as well grafted onto the agarose matrix have been analyzed and compared. Batch and dynamic (column) experiments have been performed to measure the adsorption equilibrium of proteins and to quantify the phase transition process. As model proteins lysozyme, lactoferrin, α-chymotrypsinogen A and ovalbumin have been used. The adsorption process was found to be governed by ionic interactions between the negatively charged surface of resin and the protein, which enabled separation of proteins differing in electrostatic charge. The interactions enhanced with increase of temperature. Decrease of temperature facilitated desorption of proteins and reduced the salt usage in the desorption buffer. Grafted polymers exhibited markedly higher mechanical stability and, however, weaker temperature response compared to the ungrafted ones.

Adsorption behaviour of sugars versus their activity in single and multicomponent liquid solutions.

In this work the activity of three carbohydrates (sucrose, glucose and fructose) in highly concentrated aqueous solutions was studied along with its effect on the adsorption behaviour of the investigated compounds. Activities of individual sugars in aqueous solutions of single solute as well as in binary mixtures were quantified on the basis of solubility properties. Solid-liquid equilibria of sugars were correlated with the NRTL (nonrandom, two liquid) model of activity coefficient formulation. Activities of individual sugars were incorporated into the single component adsorption isotherm model, which reproduced accurately the course of the adsorption equilibria of sugars in aqueous solutions obtained experimentally in previous work using an ion-exchange resin. Activities of sugars determined in binary solute systems along with the single component isotherms were used to predict competitive adsorption equilibria. To calculate adsorbed phase concentrations of individual sugars in binary mixtures the adsorbed solution theory was adopted. The isotherm shapes calculated were compared to the data of competitive adsorption from the former study and found to be able to describe these experimental results.

Behavior of adsorbed and fluid phases versus retention properties of amino acids on the teicoplanin chiral selector.

The relationship between adsorption equilibria of two amino acids, i.e., l,d-threonine and l,d-methionine on the teicoplanin chiral selector and their phase behavior has been analyzed. The experimental and numerical methods have been proposed to determine activity coefficients of amino acids in different solvent systems. The procedure was based on the analysis of solubility properties of the amino acids in aqueous solutions of methanol, ethanol and propanol-2-ol used as the mobile phases in chromatographic elution. The solubility measured in mixed alcohol-water solutions was correlated with the non-random-two-liquid (NRTL) model for the activity coefficients. The values of activity coefficients were incorporated into the adsorption isotherm equation, which allowed the analysis of retention properties of the amino acids versus their fluid phase behavior. For the investigation the experimental data of adsorption equilibria of amino acids as well as of the mobile phase constituents acquired in a previous work were exploited. The composition of both the mobile and the adsorbed phases was found to affect the retention properties of the amino acids. For water-rich mobile phases the activity in the adsorbed phase determined the retention mechanism, while for the alcohol-rich systems activity in the mobile phase was predominant.

Deformation of gradient shape as a result of preferential adsorption of solvents in mixed mobile phases.

Gradient elution has been studied in typical normal and reversed-phase systems. Deformations of gradient profiles have been evidenced as a result of preferential adsorption of modifiers of the mobile phase. This phenomenon was pronounced in the normal-phase system, for which gradient profiles deviated significantly from those programmed. This influenced the retention and shapes of band profiles of the eluting solute. Hence, in order to predict gradient propagation correctly the adsorption equilibrium of modifiers has been quantified. Moreover, at low modifier content, deformations of band profiles of the solute has been registered as a result of the competitive adsorption in the system solute-modifier. This effect has been predicted by a competitive adsorption model. For the reversed-phase systems the influence of the modifier adsorption on gradient propagation was insignificant for typical mobile phases investigated. Therefore, the work has been focused on gradient predictions in the normal-phase system.

Effect of temperature on competitive adsorption of the solute and the organic solvent in reversed-phase liquid chromatography.

In analysis of the temperature effect on chromatographic separations the influence of the adsorption of organic solvent on the retention properties of solute is generally not taken into account. In fact, adsorption behavior of solutes is strongly affected by competitive adsorption of organic solvents, which is temperature dependent. In this work changes of adsorption equilibrium of an organic solvent as well as a solute with temperature have been analyzed. Data of the excess adsorption of methanol from aqueous solutions on octadecyl-bonded silica have been acquired at different temperature. Experiments have been performed over a relatively narrow temperature range corresponding to typical chromatographic conditions, i.e., 10-50 degrees C. The competitive adsorption equilibria of model solutes (i.e., two homologous compounds: cyclopentanone and cyclohexanone) have been measured at different temperature and composition of the mobile phase. Temperature alterations to the retention properties were found to result from combined effects of changes in adsorption behavior of the organic solvent and of the solute. The influence of temperature on the separation selectivity has been considered.