Modeling electrostatic exclusion effects during ion exchange chromatography of monoclonal antibodies

Abstract

Recent experimental studies have shown a reduction in dynamic-binding capacity for both monoclonal antibodies and antigen-binding fragments at very low conductivity, conditions that should generate the greatest electrostatic attraction. This behavior has been attributed to the steric and electrostatic exclusion of the charged protein from the entrance of the resin pores. This manuscript presents a quantitative mathematical description of this phenomenon. The protein partition coefficient was evaluated using models for the partitioning of a charged sphere into a charged cylindrical pore, with the pore size distribution evaluated by inverse size exclusion chromatography. The results were in very good agreement with experimental data for batch protein uptake and dynamic-binding capacity over a range of pH and conductivity. This theoretical framework provides important insights into the behavior of ion exchange chromatography for protein purification.

Characterization of cross-linked cellulosic ion-exchange adsorbents: 1. Structural properties

“…Apart from steric exclusion effects of the kind measured by ISEC, there may also be electrostatic exclusion effects for proteins [24,25]. However, small ions may also be excluded due to the Donnan equilibrium effect [14] within a 3-D volume bearing fixed charges; the effect is apparent at ambient salt concentrations lower than the local volumetric density of fixed charge.…”

Protein adsorption and transport in polymer-functionalized ion-exchangers

“…Since the static capacity generally decreases with increasing salt, the DBC may be expected to do the same, but several reports [44,48,58,63–66] have demonstrated an initial increase in DBC with increasing salt concentration at low salt, leading up to a well-defined maximum and a subsequent decrease. This form of response may be seen on both conventional and polymer-functionalized media for certain proteins under appropriate conditions, but where direct comparisons are available [44,48,64], the maxima appear higher and more pronounced for the polymer-functionalized media, although the DBCs drop off more rapidly with increasing salt beyond the maxima in some cases.…”

“…More recent measurements using dextran-functionalized agarose ion-exchange media show greater partitioning of both charged and uncharged dextran probes of intermediate size with increasing salt, but little effect of salt if both the immobilized and probe dextrans are uncharged [26,58]. These results were interpreted differently, however.…”

“…This leads to a conclusion that increasing salt screens intrachain repulsion that at low salt concentrations causes expansion of the charged dextran layer, reducing the effective pore space [26]. However, an alternative argument is that even a neutral probe can interact electrostatically with a charged surface due to its displacement of the electrical double layer, with the resulting analysis [59] leading to the conclusion that the thickness of the immobilized charged dextran layer is in fact relatively unaffected by the salt concentration [58]. A questionable element of this argument is that the theory on which it is based [59] assumes that the interacting probe and surface are electrically impermeable solids, whereas in this case they are in fact polymers filled with high-dielectric water and may also admit salts that can screen electrostatic interactions.…”

“…One is the electrostatic repulsion between adsorbed and diffusing protein molecules, which results in an electrostatic exclusion effect [58,63,64]. Given the nature and range of electrostatic repulsion, this effect is likely to be important especially for large molecules, narrow pores and low salt concentrations.…”