Influence of protein and stationary phase properties on protein-matrix-interaction in cation exchange chromatography.
ABSTRACT 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.
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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.Journal of Separation Science 01/2014; 37(1-2):5-13. · 2.59 Impact Factor
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ABSTRACT: The mechanisms behind protein PEGylation are complex and dictated by the structure of the protein reactant. Hence, it is difficult to design a reaction process which can produce the desired PEGylated form at high yield. Likewise, efficient purification processes following protein PEGylation must be constructed on an ad hoc basis for each product. The retention and binding mechanisms driving electrostatic interaction-based chromatography (ion-exchange chromatography) of PEGylated proteins (randomly PEGylated lysozyme and mono-PEGylated bovine serum albumin) were investigated, based on our previously developed model Chem. Eng. Technol. 2005, 28, 1387-1393. PEGylation of each protein resulted in a shift to a smaller elution volume compared to the unmodified molecule, but did not affect the number of binding sites appreciably. The shift of the retention volume of PEGylated proteins correlated with the calculated thickness of PEG layer around the protein molecule. Random PEGylation was carried out on a column (solid-phase PEGylation) and the PEGylated proteins were separated on the same column. Solid-phase PEGylation inhibited the production of multi-PEGylated forms and resulted in a relatively low yield of selective mono-PEGylated form. Pore diffusion may play an important role in solid-phase PEGylation. These results suggest the possibility of a reaction and purification process development based on the mechanistic model for PEGylated proteins on ion exchange chromatography.Biotechnology Journal 06/2013; · 3.71 Impact Factor
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ABSTRACT: Previously, we studied bovine serum albumin (BSA) uptake to poly(ethylenimine) (PEI)-grafted Sepharose resins, and an ionic capacity (IC) range (600-740mmol/L) for steep increases of both protein capacity (qm) and effective pore diffusion coefficient (De) was found. In this work, seven PEI-grafted Sepharose FF resins at IC range of 270-1030mmol/L were synthesized to investigate the effect of protein properties on the adsorption and uptake kinetics using BSA and γ-globulin as two model proteins. For BSA, the change trends of qm and De values with IC were well consistent with the previous results. For γ-globulin, the qm values increased slowly till reaching a maximum value at IC=560mmol/L and then decreased rapidly at IC>560mol/L. The De values nearly kept unchanged at low ICs (IC<460mmol/L), and increased steeply at IC>460mmol/L till reaching a maximum at 680mmol/L (De/D0=0.48±0.01). After that increase, the De values for γ-globulin dropped quickly at IC>680mol/L, which was not observed for BSA. It is interesting to note that in the narrow IC range of 460-680mmol/L, the De values of γ-globulin increased dramatically for more than four folds. Moreover, it is notable that the IC range where the hopping of De values occurred for γ-globulin was earlier than that for BSA (460 vs. 560mmol/L). The earlier hopping of γ-globulin uptake rate was attributed to its larger size and less net charge, which facilitated the happenings of the "chain delivery" effect. The quick drops of both qm and De values for γ-globulin at IC>680mmol/L were considered due to its large size, which led to the significant decrease of its effective pore volume. The results indicate that both PEI layer and protein size played important roles in protein adsorption to PEI-grafted resins, and further prove the "chain delivery" effect did contributed significantly to the uptake rate hopping in the PEI-grafted resins. This work could also help the design and selection of resins based on protein characteristics and benefit optimization of practical chromatographic processes for therapeutic proteins with PEI-grafted anion exchangers.Journal of Chromatography A 03/2014; · 4.61 Impact Factor