Modeling of salt and pH gradient elution in ion-exchange chromatography

Abstract

The separation of proteins by internally and externally generated pH gradients in chromatofocusing on ion-exchange columns is a well-established analytical method with a large number of applications. In this work, a stoichiometric displacement model was used to describe the retention behavior of lysozyme on SP Sepharose FF and a monoclonal antibody on Fractogel SO3 (S) in linear salt and pH gradient elution. The pH dependence of the binding charge B in the linear gradient elution model is introduced using a protein net charge model, while the pH dependence of the equilibrium constant is based on a thermodynamic approach. The model parameter and pH dependences are calculated from linear salt gradient elutions at different pH values as well as from linear pH gradient elutions at different fixed salt concentrations. The application of the model for the well-characterized protein lysozyme resulted in almost identical model parameters based on either linear salt or pH gradient elution data. For the antibody, only the approach based on linear pH gradients is feasible because of the limited pH range useful for salt gradient elution. The application of the model for the separation of an acid variant of the antibody from the major monomeric form is discussed.

Purification of common light chain IgG-like bispecific antibodies using highly linear pH gradients

“…Examples of such buffer systems have recently been described by a group specializing in the modeling of pH gradients for IEX. 38,39 An additional consideration for preparative scale purifications is the stability of the protein at the starting pH of the separation, since the protein mixture is usually being loaded onto the column in a buffer of the same pH. Although we did not encounter any stability issues at the high end of the pH range (the loading pH for AEX), we found that, especially for BsAbs made up of IgG4 parents, a starting pH of 4.0 led to significant amounts of aggregation (results not shown).…”

Modeling of dual gradient elution in ion exchange and mixed-mode chromatography

“…Schmidt et al [31] adapted Eqs. (1) and (2) to describe protein binding in IEC using linear pH gradient, which is given by the following expression:…”

“…We also demonstrate that Yamamoto's LGE model [27][28][29] which is widely used to describe protein retention with linear isotherm in IEC using salt [30] or pH gradient [31] can also describe protein-ligand binding in mixed-mode chromatography. By using Mollerup's thermodynamic definition of Gibbs free energy, the pH dependency of the equilibrium constant is introduced to the stoichiometric displacement (SD)-based-LGE model [22,32].…”

“…By using Mollerup's thermodynamic definition of Gibbs free energy, the pH dependency of the equilibrium constant is introduced to the stoichiometric displacement (SD)-based-LGE model [22,32]. The pH dependency of the stoichiometric number of electrostatic interactions is expressed as a function of protein net charge correlated to the number of amine groups (N Lys , N Arg and N N-term ), carboxyl groups (N Carboxyl ), cysteines (N Cys ), tyrosines (N Tyr ) and histidines (N His ) which are involved or affect protein-ligand bindings [31,33,34]. Furthermore, the LGE model is extended to describe protein separation using dual gradient system in which the salt concentration and pH change simultaneously.…”

Selective targeting of the IL23 pathway: Generation and characterization of a novel high-affinity humanized anti-IL23A antibody

“…59 IgG1 antibodies for the selected humanized and optimized clones were expressed initially in HEK293 cells transiently and then in NS0 cells as stable transfectants and purified using standard methods with modifications. [60][61][62] The purified samples were then submitted for biophysical profiling, functional testing, and other studies.…”