Simulation model for overloaded monoclonal antibody variants separations in ion-exchange chromatography.

A model was developed for the design of a monoclonal antibody charge variants separation process based on ion-exchange chromatography. In order to account for a broad range of operating conditions in the simulations, an explicit pH and salt concentration dependence has been included in the Langmuir adsorption isotherm. The reliability of this model was tested using experimental chromatographic retention times as well as information about the structural characteristics of the different charge variants, e.g. C-terminal lysine groups and deamidated groups. Next, overloaded isocratic elutions at various pH and salt concentrations have been performed to determine the saturation capacity of the ion-exchanger. Furthermore, the column simulation model was applied for the prediction of monoclonal antibody variants separations with both pH and salt gradient elutions. A good prediction of the elution times and peak shapes was observed, even though none of the model parameters was adjusted to fit the experimental data. The trends in the separation performance obtained through the simulations were generally sufficient to identify the most promising operating conditions. The predictive column simulation model thus developed in this work, including a set of parameters determined through specific independent experiments, was experimentally validated and offers a useful basis for a rational optimization of monoclonal antibody variants separation processes on ion-exchange chromatography.

Electrostatic model for protein adsorption in ion-exchange chromatography and application to monoclonal antibodies, lysozyme and chymotrypsinogen A.

A model for the adsorption equilibrium of proteins in ion-exchange chromatography explicitly accounting for the effect of pH and salt concentration in the limit of highly diluted systems was developed. It is based on the use of DLVO theory to estimate the electrostatic interactions between the charged surface of the ion-exchanger and the proteins. The corresponding charge distributions were evaluated as a function of pH and salt concentration using a molecular approach. The model was verified for the adsorption equilibrium of lysozyme, chymotrypsinogen A and four industrial monoclonal antibodies on two strong cation-exchangers. The adsorption equilibrium constants of these proteins were determined experimentally at various pH values and salt concentrations and the model was fitted with a good agreement using three adjustable parameters for each protein in the whole range of experimental conditions. Despite the simplifications of the model regarding the geometry of the protein-ion-exchanger system, the physical meaning of the parameters was retained.

Peptide pore accessibility in reversed-phase chromatography.

The effect of salt or peptide concentration on peptide porosity (i.e. the porosity accessible to a given peptide) is investigated on six different reversed-phase stationary phases. The peptide porosity is found to increase with the local concentration of negative charges following a saturation-type function within the same porosity boundaries for both cases. This can induce the formation of anti-Langmuirian peaks in non-adsorbing conditions since the local increase of the ionic strength due to the peptide concentration increases the porosity accessible to the peptide. This behavior can be well reproduced by the ideal model of chromatography assuming non-constant porosity. The acetonitrile adsorption isotherm was also measured on all the considered reversed-phase stationary phases. A comparison between the stationary phases shows a correlation between the amount of acetonitrile accumulated in the pores and the reduced pore accessibility for the peptide.

Chromatographic separation of three monoclonal antibody variants using multicolumn countercurrent solvent gradient purification (MCSGP).

Multicolumn countercurrent solvent gradient purification (MCSGP) is a continuous chromatographic process developed in recent years (Aumann and Morbidelli, 2007a; Aumann et al., 2007) that is particularly suited for applications in the field of bioseparations. Like batch chromatography, MCSGP is suitable for three-fraction chromatographic separations and able to perform solvent gradients but it is superior in terms of solvent consumption, yield, purity, and productivity due to the countercurrent movement of the liquid and the solid phases. In this work, the MCSGP process is applied to the separation of three monoclonal antibody variants on a conventional preparative cation exchange resin. The experimental process performance was compared to simulations based on a lumped kinetic model. Yield and purity values of the target variant of 93%, respectively were obtained experimentally. The batch reference process was clearly outperformed by the MCSGP process.

Role of recycling in improving the performance of chromatographic solvent gradient purifications.

With significant advancement in the upstream processing technology, downstream processing of large bio-molecules is becoming the bottle-neck in the production chain. To face this challenge, design and development of efficient separation processes has become crucial. As a step towards boosting the performance of a chromatographic separation process through improved design, we investigated the potential of recycling as a process option. The most important advantage of recycling is that it can be implemented in an existing batch system without any major investment and consultation. Although impure products are recycled in industries, it is done as additional batch, and only then, when the recoverable product is valuable enough to surpass the loss of productivity in running the additional batches. In our study, on the other hand, it was found that a well-designed recycle can not only improve the yield, but also the productivity of a multi-component purification. A series of multiobjective optimization studies were carried out on multi-component separation to comprehend the role of recycling with reference to an industrially relevant problem, i.e. the chromatographic purification step of the production process of calcitonin.

Adsorption of monoclonal antibody variants on analytical cation-exchange resin.

Monoclonal antibody (MAb) variants differing by one or two C-terminal lysine residues can be separated by cation-exchange chromatography due to the difference in their charge distribution. The adsorption of the three MAb variants on a weak cation-exchange resin was characterized using directly the raw mixture in spite of the presence of some impurities. The effects of both, pH and eluent salt concentration, on the adsorption isotherm were investigated. Under certain experimental conditions distorted peak shapes and even peak doubling for single variant injections were obtained, in addition to unexpectedly long retention times. These observations were explained based on equilibrium theory. The separation of the MAb variants was designed for an isocratic and a linear salt gradient operation. The corresponding optimal values of pH and salt concentration were determined. The use of salt gradients not only allows reducing the process time and increasing enrichment of the variants, but also leads to some loss in purity. A baseline separation could be obtained under isocratic and strongly adsorbing conditions at pH 6.3. A lumped kinetic model and a procedure for estimating the corresponding parameters were developed and validated by comparison with experimental elution chromatograms in a wide range of operating conditions.

A continuous, counter-current multi-column chromatographic process incorporating modifier gradients for ternary separations.

The existing literature of continuous chromatographic process indicates the scarcity of processes which allow the continuous separation of a ternary mixture and which also exploit the power of modifier gradients and counter-currency. A new process filling this gap is presented and analyzed using an equilibrium theory model and linear isotherms. The analysis leads to the explicit formulation of an operating region, where 100% purity and yield can be obtained. A normalized flow rate ratio is introduced, which allows an easy description and understanding of the process behavior. The gained insights are verified using a detailed simulation model, where the influence of changes in the normalized flow rate ratios on the unit performance is investigated.

Experimental verification of sample-solvent induced modifier-solute peak interactions in biochromatography.

In a previous theoretical analysis based on equilibrium theory it has been shown how differences in the sample and elution modifier concentrations can lead to unexpected behavior of the solute eluted peaks such as retention time distortion, peak deformation and peak doubling. All these features are verified experimentally in this work using the polypeptide calcitonin and a variant of a specific monoclonal antibody as chromatographic model systems. For both experimental systems, the retention time distortion can be predicted with high accuracy by the solution of the equilibrium theory model. For the polypeptide, the predictions from the theory about the occurrence of peak deformation and double peaks has been successfully verified by a series of tailored experiments with positive as well as negative modifier perturbations.

Analysis of sample-solvent induced modifier-solute peak interactions in biochromatography using equilibrium theory and detailed simulations.

Batch liquid chromatographic columns are often equilibrated with an eluent stream being a mixture of inert compounds and so-called modifiers. The sample injected into the eluent stream usually consists of the solutes to be separated and of a mixture of the same solvents as in the eluent but in general with different concentration values. This results in two groups of peaks moving along the column: the solute peaks and the modifier pertubations. If the adsorptivity of the solute depends strongly on the modifier, as it is often the case in biochromatography, the interference between the two groups of peaks leads to peculiar phenomena like double peaks, split peaks, distorted peaks with anti-Langmurian shape, etc. In this work, these phenomena are analyzed based on an analytical solution of the equilibrium theory model and the results are compared with detailed simulations and experimental data. It is shown that the qualitative behavior is well predicted in the frame of equilibrium theory and general guidelines how to avoid these kinds of interactions are developed.