Ion exchange chromatography of proteins—predictions of elution curves and operating conditions. II. Experimental verification

Abstract

The applicability and validity of the model developed in Part I were confirmed experimentally. In this article, various proteins were eluted both by stepwise and linear gradient elution on DEAE ion exchangers under a variety of experimental conditions. Adsorption isotherms were measured as a function of ionic strength in batch experiments. The moment method was employed for the determination of various parameters such as the gel-phase diffusion coefficient and the longitudinal dispersion coefficient. By use of these parameters and the experimentally measured ionic strength of the peak position, the number of plates was determined according to the method described in Part I. Theoretical elution curves were calculated with the experimentally measured adsorption equilibria and the number of plates. Good agreement was observed between theory an experiments. Various factors affecting the separation were investigated. It was found that the effect of the number of plates for salts, N'(p), was negligible except the case of stepwise elution of high ionic strength buffer. When elution curves were symmetrical, the widths of the elution curves were inversely proportional to the square root of the number of plates of proteins, N(p), as in other chromatographic techniques. A simple graphical method for prediction of the peak position in linear gradient elution described in Part I was found applicable when the elution curves were symmetrical. A useful correlation of prediction of the peak width in a linear gradient elution was proposed on the basis of the approximate solution derived in Part I of this study. This graphical method and correlation permit easy prediction of the peak position and peak width in linear gradient elution in the case of symmetrical elution curves.

Rate parameters and gradient correlations for gradient‐elution chromatography

“…This method is simpler and less time-consuming than the isotherm method used by Yamamoto et al (1983b). The protein adsorption isotherm in the model could be either linear or nonlinear, depending on the concentration of the protein solution.…”

“…This method is simpler and less time-consuming than the isotherm method used by Yamamoto et al (1983b). P-Lactoglobulin A (0.025%)…”

“…The diameter of the adsorbent particle is 0.011 cm. It was chosen for the present work because it was used before for p-lactoglobulin intraparticle diffusion studies (Boyer and Hsu, 1992) and gradient-elution studies (Yamamoto et al, 1983b), where adsorption isotherms and intraparticle diffusivity correlation were determined. Adsorption for both proteins is linear as long as the protein concentrations are less than 0.03% (weight to volume, Yamamoto et al, 1983b).…”

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

“…The linear gradient elution model developed by Yamamoto delivers data for the prediction of protein elution behavior, as well as information about stationary phase properties and electrostatic and non-electrostatic proteinmatrix-interactions. 17,18 Linear gradient elutions were performed and analysed as described by Ishihara and Yamamoto to obtain information about the strength of protein-matrix-interactions. 16 Other important properties of chromatographic media are adsorption capacities and mass transfer characteristics.…”

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“…Results from the previous investigation for were found to be 0.325 and 0.362 for modules 1 and 2, respectively . For the estimation of D ieff , Yamamoto et al (1983aYamamoto et al ( , 1983b found that (D ieff /u i ) is constant regardless of the flow rate and type of gel particles and solutes, and is a unique function of the particle size of the gel. Here, u i is the interstitial velocity in the packed bed.…”