pH transitions, protein separations, and mass transfer in chromatography columns containg weak ion exchange groups

The use of ion exchange columns containing weak acid or weak base groups provides both challenges and opportunities. The challenges arise from the complex pH transitions that occur in response to changes in the mobile phase composition. If not understood and accounted for, such transitions may result in unreliable or inefficient processes. The opportunities, on the other hand, arise from the possibility of utilizing such pH transitions to obtain the separation of complex protein mixtures.

This dissertation presents a comprehensive local equilibrium model to predict the magnitude and duration of these pH transients. The model equations are solved by the method of characteristics and by numerical simulations using an equilibrium-dispersive model. Applications are considered for three different systems: (i) pH transients in response to changes in salt concentration with constant buffer composition; (ii) pH gradients induced by positive pH steps with weak cation exchangers; and (iii) pH transients induced in response to negative pH steps with anion exchangers. In all three cases, the model, predictions are in excellent agreement with experimental results providing a robust way to select optimum buffers and operating conditions for each application. This dissertation also explores use of induced pH gradients with weak exchangers for the separation of closely related proteins including the separation of mixtures of ovalbumin, albumin, and transferrin with a weak cation exchanger and the separation of protein charge variants with different weak anion exchangers. In both cases, the effects of buffer composition and concentration, pH, ionic strength, flow rate, and protein loading are investigated systematically.

Lastly, this dissertation considers protein mass transfer in resins containing different amounts of weak acid groups. The effects are generally small, in this case, indicating that protein mass transfer, which is dominated by pore diffusion mechanisms, is largely unaffected by the resin's surface chemistry. Overall, this dissertation provides the tools and experimental analyses needed for a comprehensive assessment of challenges and opportunities arising with the use of weak ion exchangers in protein downstream processing.

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