Application of linear pH gradients for the modeling of ion exchange chromatography: Separation of monoclonal antibody monomer from aggregates

Abstract

The mobile phase pH is a key parameter of every ion exchange chromatography process. However, mechanistic insights into the pH influence on the ion exchange chromatography equilibrium are rare. This work describes a mechanistic model capturing salt and pH influence in ion exchange chromatography. The pH dependence of the characteristic protein charge and the equilibrium constant is introduced to the steric mass action model based on a protein net charge model considering the number of amino acids interacting with the stationary phase. This allows the description of the adsorption equilibrium of the chromatographed proteins as a function of pH. The model parameters were determined for a monoclonal antibody monomer, dimer, and a higher aggregated species based on a manageable set of pH gradient experiments. Without further modification of the model parameters the transfer to salt gradient elution at fixed pH is demonstrated. A lumped rate model was used to predict the separation of the monoclonal antibody monomer/aggregate mixture in pH gradient elution and for a pH step elution procedure-also at increased protein loadings up to 48 g/L packed resin. The presented model combines both salt and pH influence and may be useful for the development and deeper understanding of an ion exchange chromatography separation.

Evaluation of differences between dual salt‐pH gradient elution and mono gradient elution using a thermodynamic model: Simultaneous separation of six monoclonal antibody charge and size variants on preparative‐scale ion exchange chromatographic resin

“… All parameters were given by manufacturer. Other parameters were determined according to Kluters et al…”

“…In the linear range of the isotherm (i.e. at low protein loading), the distribution coefficient can be calculated as the ratio between adsorbed protein concentration in the stationary phase and protein concentration in the mobile phase , i.e., . The characteristic binding charge of the protein is a function of pH which is defined by Schmidt et al based on the protein net charge model as: where the number of acidic amino acids is ( , ) and the number of each basic amino acid is ( , , , ) involved in protein‐ligand binding.…”

“…Recently, Kluters et al and Lee et al have described a mathematical model based on Yamamoto's LGE model which can be used to investigate the influence of salt and pH on the characteristic binding charge and the equilibrium constant in IEC. This model when combined with a lumped rate chromatography model can be used to design protein separation processes based on the respective simulated salt and pH gradient elution curves …”

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).…”

Systematic interpolation method predicts protein chromatographic elution with salt gradients, pH gradients and combined salt/pH gradients

“…Fitting the number of each charged residue in this set to chromatographic data provided a functional description of pH and salt gradient elution. Kluters et al extended the work of Schmidt and coworkers to overloaded conditions using the SMA model but did not validate high protein load conditions with a combined pH‐salt gradient. In yet another approach, Vetter et al fitted the SMA model to isotherm data describing mAb binding to a CEX resin as a function of protein and salt concentration and then correlated the dependence of the protein binding charge on pH using a linear empirical relationship.…”