Christian Frech

Cation-exchange chromatography of monoclonal antibodies: Characterisation of a novel stationary phase designed for production-scale purification

A novel cation-exchange resin, Eshmuno™ S, was compared to Fractogel® SO3- (M) and Toyopearl GigaCap S-650M. The stationary phases have different base matrices, and carry specific types of polymeric surface modifications. Three monoclonal antibodies (mAbs) were used as model proteins to characterize these chromatographic resins. Results from gradient elutions, stirred batch adsorptions and confocal laser scanning microscopic investigations were used to elucidate binding behaviour of mAbs onto Eshmuno™ S and Fractogel® SO3- and the corresponding transport mechanisms on these two resins. The number of charges involved in mAb binding for Eshmuno™ S is lower than for Fractogel® SO3-, indicating a slightly weaker electrostatic interaction. Kinetics from batch uptake experiments are compared to kinetic data obtained from confocal laser scanning microscopy images. Both experimental approaches show an accelerated protein adsorption for the novel stationary phase. The influence of pH, salt concentrations and residence times on dynamic binding capacities was determined. A higher dynamic binding capacity for Eshmuno™ S over a wider range of pH values and residence times was found compared to Fractogel® SO3- and Toyopearl GigaCap S-650M. The capture of antibodies from cell culture supernatant, as well as post-protein A eluates, were analyzed with respect to their host cell protein (hcp) removal capabilities. Comparable or even better hcp clearance was observed at much higher protein loading for Eshmuno™ S than Fractogel® SO3- or Toyopearl GigaCap S-650M.

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

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.

Influence of protein and stationary phase properties on protein-matrix-interaction in cation exchange chromatography.

A large number of different stationary phases for ion-exchange chromatography from different manufacturers are available, which vary significantly in a number of chemical and physical properties. As a consequence, binding mechanisms may be different as well. In the work reported here, the retention data of model proteins (lysozyme, cytochrome c and two monoclonal antibodies) were determined for nine commercially available cation-exchange adsorbents. The linear gradient elution model in combination with a thermodynamic approach was used to analyse the characteristic parameters of the protein-stationary phase-interactions. Based on the pH dependency of the characteristic charge and the equilibrium constant for binding the differences between the standard Gibbs energies in the adsorbed and the solute state for the protein ΔG(P)° and the salt ΔG(S)° were calculated. The characteristic charge B of the proteins strongly depends on the molecular mass of the protein. For small proteins like lysozyme there is almost no influence of the stationary phase chemistry on B, while for the Mabs the surface modification strongly influences the B value. Surface extenders or tentacles usually increase the B values. The variation of the characteristic charge of the MABs is more pronounced the lower the pH value of the mobile phase is, i.e. the higher the negative net charge of the protein is. The standard Gibbs energy changes for the proteins ΔG(P)° are higher for the Mabs compared to lysozyme and more strongly depend on the stationary phase properties. Surface modified resins usually show higher ΔG(P)° and higher B values. A correlation between ΔG(P)° and B is not observed, indicating that non-electrostatic interactions as well as entropic factors are important for ΔG(P)° while for the B values the accessibility of binding sites on the protein surface is most important.

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

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.

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

Protein retention using dual gradient elution in ion exchange- and mixed-mode chromatography can be modeled using the combination of a modified Yamamotos LGE model and a conversion term to correlate the elution salt concentration and pH at any given gradient slope. Incorporation of the pH dependence of the binding charges into the model also provides some insights on the dual effects of salt and pH in protein-ligand interaction. The fitted thermodynamic parameters (ΔGP(0)/RT, ΔGS(0)/RT, number of charged amino acids involved in binding) of the dual gradient elution data using lysozyme and mAbs on SP Sepharose(®) FF, Eshmuno(®) HCX, and Capto(®) MMC ImpRes were consistent to the results of mono gradient data. This gives rise to an approach to perform thermodynamic modeling of protein retention in ion exchange- and mixed-mode chromatography by combining both salt and pH gradient into a single run of dual gradient elution which will increase time and cost efficiency. The dual gradients used in this study encompassed a wide range of pH (4-8) and NaCl concentrations (0-1M). Curve fits showed that ΔGP(0)/RT is protein type and ligand dependent. ΔGS(0)/RT is strongly dependent on the stationary phase but not the protein. For mAb04 on mixed-mode resin Capto(®) MMC, ΔGS(0)/RT is 5-6 times higher than the result reported for the same protein on cation exchanger Fractogel(®) EMD SO3(-) (S).

Solvent modulated linear pH gradient elution for the purification of conventional and bispecific antibodies: Modeling and application

Classical ion-exchange chromatography using a linear salt gradient to elute the adsorbed protein at fixed pH is the most common method to separate product-related impurities during downstream processing of biopharmaceuticals. Linear pH gradient elution provides a useful alternative by separating proteins in a linear pH gradient at fixed salt concentration. Although linear pH gradient elution provides excellent selectivity, it is rarely encountered in industrial purification processes. Here, a stoichiometric displacement model is used to characterize pH gradient elution based on simple linear gradient elution experiments. Protein retention behavior is described with respect to the pH dependencies of the characteristic binding charge and the equilibrium constant of the ion exchange reaction. Furthermore, the influence of solvent composition using PEG as a mobile phase modifier is investigated. Validity and applicability of the model are demonstrated for the purification of a conventional monoclonal antibody from soluble aggregates and for a novel bispecific antibody format containing a unique product-related impurity profile. pH step elution protocols are derived from model calculations without further optimization experiments necessary.

Mechanism of improved antibody aggregate separation in polyethylene glycol-modulated cation exchange chromatography.

Ion-exchange chromatography is used in biopharmaceutical downstream processes to reduce product-related impurity levels. Because protein aggregate levels can be considered as a critical quality attribute, the removal of aggregated protein species is of primary importance. The addition of polyethylene glycol (PEG) to the mobile phase in ion-exchange chromatography was found to significantly improve the chromatographic separation of monomers from aggregates. In this work, linear gradient elution experiments with monomeric and aggregated samples of a monoclonal antibody were performed on a strong cation exchange resin at different PEG concentrations to investigate the underlying effects responsible for the observed selectivity improvement. PEG is well known to be excluded from a surface layer volume around the protein and the stationary phase; thus, enhancing adsorption of the preferentially hydrated protein to the hydrated stationary phase. The exclusion volume depends on the accessible surface area of the protein leading to a stronger influence of PEG on larger protein species and thus an improved separation of monomer and aggregates. This hypothesis could be consolidated comparing the distribution equilibrium in PEG solution to that in water by calculating equilibrium constants and transfer free energies using the chromatographic data from the linear gradient elution experiments performed at different pH values.

Solvent modulation strategy for superior antibody monomer/aggregate separation in cation exchange chromatography.

Cation exchange chromatography (CEX) is an integral part of many downstream processes for monoclonal antibodies (mAbs). However, in some cases CEX methods with standard mobile phase conditions do not lead to a sufficient removal of soluble antibody aggregates. The addition of neutral polymers such as polyethylene glycol (PEG) to the mobile phase can improve the separation of proteins in IEC remarkably. The applicability of this solvent modulation technique is limited by protein precipitation at higher PEG concentrations. To overcome this limitation solubility enhancers like polyols and amino acids can be added to the mobile phase. These additives are known to inhibit PEG-induced protein precipitation in solution. This new solvent modulation strategy was tested with three different mAbs on two different CEX resins in the presence of PEG in combination with various solubility enhancers. In order to assess the general applicability of this method, mAbs were selected that show major differences with respect to their sensitivity to PEG-induced precipitation and monomer/aggregate resolution performance that is achieved by CEX under standard conditions. For all three mAbs precipitation could be prevented without elimination of the positive PEG-effect. The addition of solubility enhancers gives access to improved separation at elevated PEG concentrations and high protein loadings without running into precipitation issues. Our data indicate that this method is generically applicable and leads to a superior antibody monomer/aggregate separation.

Thermodynamic modeling of protein retention in mixed-mode chromatography: An extended model for isocratic and dual gradient elution chromatography.

An extended model is developed to describe protein retention in mixed-mode chromatography based on thermodynamic principles. Special features are the incorporation of pH dependence of the ionic interaction on a mixed-mode resin and the addition of a water term into the model which enables one to describe the total number of water molecules released at the hydrophobic interfaces upon protein-ligand binding. Examples are presented on how to determine the model parameters using isocratic elution chromatography. Four mixed-mode anion-exchanger prototype resins with different surface chemistries and ligand densities were tested using isocratic elution of two monoclonal antibodies at different pH values (7-10) and encompassed a wide range of NaCl concentrations (0-5M). U-shape mixed-mode retention curves were observed for all four resins. By taking into account of the deprotonation and protonation of the weak cationic functional groups in these mixed-mode anion-exchanger prototype resins, conditions which favor protein-ligand binding via mixed-mode strong cationic ligands as well as conditions which favor protein-ligand binding via both mixed-mode strong cationic ligands and non-hydrophobic weak cationic ligands were identified. The changes in the retention curves with pH, salt, protein, and ligand can be described very well by the extended model using meaningful thermodynamic parameters like Gibbs energy, number of ionic and hydrophobic interactions, total number of released water molecules as well as modulator interaction constant. Furthermore, the fitted model parameters based on isocratic elution data can also be used to predict protein retention in dual salt-pH gradient elution chromatography.

Modeling and simulation of protein elution in linear pH and salt gradients on weak, strong and mixed cation exchange resins applying an extended Donnan ion exchange model

Process development and characterization based on mathematic modeling provides several advantages and has been applied more frequently over the last few years. In this work, a Donnan equilibrium ion exchange (DIX) model is applied for modelling and simulation of ion exchange chromatography of a monoclonal antibody in linear chromatography. Four different cation exchange resin prototypes consisting of weak, strong and mixed ligands are characterized using pH and salt gradient elution experiments applying the extended DIX model. The modelling results are compared with the results using a classic stoichiometric displacement model. The Donnan equilibrium model is able to describe all four prototype resins while the stoichiometric displacement model fails for the weak and mixed weak/strong ligands. Finally, in silico chromatogram simulations of pH and pH/salt dual gradients are performed to verify the results and to show the consistency of the developed model.