Description of Adsorption in Liquid Chromatography under Nonideal Conditions.

A thermodynamically consistent description of binary adsorption in reversed-phase chromatography is presented, accounting for thermodynamic nonidealities in the liquid and adsorbed phases. The investigated system involves the adsorbent Zorbax 300SB-C18, as well as phenetole and 4- tert-butylphenol as solutes and methanol and water as inert components forming the eluent. The description is based on adsorption isotherms, which are a function of the liquid-phase activities, to account for nonidealities in the liquid phase. Liquid-phase activities are calculated with a UNIQUAC model established in this work, based on experimental phase equilibrium data. The binary interaction in the adsorbed phase is described by the adsorbed solution theory, assuming an ideal (ideal adsorbed solution theory) or real (real adsorbed solution theory) adsorbed phase. Implementation of the established adsorption model in a chromatographic code achieves a quantitative description of experimental elution profiles, with feed compositions exploiting the entire miscible region, and involving a broad range of different eluent compositions (methanol/water). The quantitative agreement of the model and experimental data serves as a confirmation of the underlying physical (thermodynamic) concepts and of their applicability to a broad range of operating conditions.

Interconversion and chromatographic separation of carbohydrate stereoisomers on polystyrene-divinylbenzene resins.

Mutarotation of sugars causes distorted and splitted peaks in liquid chromatography. The shape of the elution profile is affected by the different anomeric and isomeric forms in solution. The rate of interconversion between the different forms relative to the propagation velocity in the column determines the extent of the distortion. This interplay of interconversion reaction and chromatographic separation was examined both experimentally and theoretically. Elution profiles resulting from pulse injections of glucose and fructose solutions at different flow rates and temperatures were analyzed both qualitatively and quantitatively. Adsorption equilibrium and reaction kinetic parameters were estimated by a simple fitting procedure, based on peak elution times and area ratios obtained from the analysis of the experimental profiles. To enhance the accuracy of the model parameters further, estimated reaction kinetic parameters were provided as an initial guess for inverse fitting to elution profiles, using a numeric mass balance model. Simulations with the numeric model, based on the enhanced parameters, allowed a very precise description of the experimental profiles. Accuracy of the fitted parameters was further confirmed through comparison with literature values. Reaction rate constants for the adsorbed phase were calculated and interpreted based on apparent rate constant values determined in this work, and on literature data for aqueous solutions.

Absence of experimental evidence of a delta-shock in the system phenetole and 4-tert-butylphenol on Zorbax 300SB-C18.

We investigate the system consisting of phenetole (PNT) and 4-tert-butylphenol (TBP) in methanol-water (63:37 v:v) on a Zorbax 300SB-C18 column by characterising single component isotherms, by performing a large number of binary experiments of different types and by describing the experiments through simulations carried out using a novel, rather powerful competitive adsorption isotherm, that we call the generalized bi-Langmuir isotherm. This system is of great interest because it was previously reported to yield a new type of transition in nonlinear chromatography, the so-called delta-shock. Such transition had been discovered earlier through a theoretical analysis and confirmed by detailed simulations. The initial aim of this work was to reach a satisfactory agreement between delta-shock experiments and corresponding numerical simulations. In the course of this work however, a number of inconsistencies in the interpretation of the previous experimental results were highlighted and explained. This led to a new experimental campaign, which is reported here and has allowed to reach two important conclusions: (1) The binary system PNT-TBP mentioned above does not exhibit a delta-shock; the spike in the UV profile, which has previously been interpreted as an experimental evidence of the delta-shock, results from liquid-liquid phase separation within the chromatographic column. (2) The same system exhibits a rather peculiar behavior in breakthrough and displacement experiments, which could be well described using the generalized bi-Langmuir isotherm.

Equilibrium theory analysis of liquid chromatography with non-constant velocity.

In liquid chromatography, adsorption and desorption lead to velocity variations within the column if the adsorbing compounds make up a high volumetric ratio of the mobile phase and if there is a substantial difference in the adsorption capacities. An equilibrium theory model for binary systems accounting for these velocity changes is derived and solved analytically for competitive Langmuir isotherms. Characteristic properties of concentration and velocity profiles predicted by the derived model are illustrated by two exemplary systems. Applicability of the model equations for the estimation of isotherm parameters from experimental data is investigated, and accurate results are obtained for systems with one adsorbing and one inert compound, as well as for systems with two adsorbing compounds.

Fast and scalable purification of a therapeutic full-length antibody based on process crystallization.

The potential of process crystallization for purification of a therapeutic monoclonal IgG1 antibody was studied. The purified antibody was crystallized in non-agitated micro-batch experiments for the first time. A direct crystallization from clarified CHO cell culture harvest was inhibited by high salt concentrations. The salt concentration of the harvest was reduced by a simple pretreatment step. The crystallization process from pretreated harvest was successfully transferred to stirred tanks and scaled-up from the mL-scale to the 1 L-scale for the first time. The crystallization yield after 24 h was 88-90%. A high purity of 98.5% was reached after a single recrystallization step. A 17-fold host cell protein reduction was achieved and DNA content was reduced below the detection limit. High biological activity of the therapeutic antibody was maintained during the crystallization, dissolving, and recrystallization steps. Crystallization was also performed with impure solutions from intermediate steps of a standard monoclonal antibody purification process. It was shown that process crystallization has a strong potential to replace Protein A chromatography. Fast dissolution of the crystals was possible. Furthermore, it was shown that crystallization can be used as a concentrating step and can replace several ultra-/diafiltration steps. Molecular modeling suggested that a negative electrostatic region with interspersed exposed hydrophobic residues on the Fv domain of this antibody is responsible for the high crystallization propensity. As a result, process crystallization, following the identification of highly crystallizable antibodies using molecular modeling tools, can be recognized as an efficient, scalable, fast, and inexpensive alternative to key steps of a standard purification process for therapeutic antibodies.