An exclusion mechanism in ion exchange chromatography

Abstract

Protein dynamic binding capacities on ion exchange resins are typically expected to decrease with increasing conductivity and decreasing protein charge. There are, however, conditions where capacity increases with increasing conductivity and decreasing protein charge. Capacity measurements on two different commercial ion exchange resins with three different monoclonal antibodies at various pH and conductivities exhibited two domains. In the first domain, the capacity unexpectedly increased with increasing conductivity and decreasing protein charge. The second domain exhibited traditional behavior. A mechanism to explain the first domain is postulated; proteins initially bind to the outer pore regions and electrostatically hinder subsequent protein transport. Such a mechanism is supported by protein capacity and confocal microscopy studies whose results suggest how knowledge of the two types of IEX behavior can be leveraged in optimizing resins and processes. ß

Modeling electrostatic exclusion effects during ion exchange chromatography of monoclonal antibodies

“…Zydney the rate of protein mass transfer into the particle at low conductivity and low pH observed by Harinarayan et al (2006) and Faude et al (2007).…”

“…Recent studies of dynamic-binding capacities for several monoclonal antibodies in SP Sepharose TM XL and SP Sepharose Fast Flow cation exchange resins showed a significant reduction in dynamic-binding capacity at very low ionic strength and low pH (Harinarayan et al, 2006) conditions that would have been expected to generate the greatest electrostatic attraction between the positively charged proteins and the negatively charged resin. Confocal scanning laser microscopy demonstrated that this reduction in dynamic-capacity was caused by a dramatic reduction in mass transfer rates within the porous cation exchange particles, with the antibodies penetrating only a few microns into the particle interior under low ionic strength conditions.…”

“…Confocal scanning laser microscopy demonstrated that this reduction in dynamic-capacity was caused by a dramatic reduction in mass transfer rates within the porous cation exchange particles, with the antibodies penetrating only a few microns into the particle interior under low ionic strength conditions. Harinarayan et al (2006) hypothesized that this reduction in dynamic-binding capacity was due to an electrostatic and steric exclusion mechanism, with the initial binding of the charged protein to the outer region of the pores hindering subsequent transport of charged proteins into the pores.…”

“…No quantitative analysis of these mechanisms was presented, nor was there any independent data for the underlying phenomena. Harinarayan et al (2006) attributed the ''exclusion mechanism'' in the cation exchange resins to a steric and electrostatic repulsion that developed because the effective charge of the cation exchanger became positive, that is, of the same polarity as the protein, at the high levels of adsorption of the positively charged antibody at the outer region of the particle. This type of ''charge reversal'' has been reported previously by Yamamoto et al (1992) for carboxymethyl Sephadex C-25 particles, with the zeta potential of the negatively charged particles becoming positive (as measured by microelectrophoresis) after adsorption of positively charged lysozyme.…”

Imaging of Fluorophores in Chromatographic Beads, Reconstruction of Radial Density Distributions and Characterisation of Protein Uptaking Processes

“…CLSM provides the visualisation of an intraparticle protein distribution (i.e., an inhomogeneous protein density) without mechanical destruction of the resin particle and explanation of absorption and diffusion mechanisms inside many chromatographic absorbents. There is a reach literature on CLSMbased chromatography, see, e.g., Ljunglöf and Hjorth (1996); Ljunglöf and Thömmes (1998); Linden et al (1999); Dziennik et al (2003); Hubbuch et al (2003); Kasche et al (2003); Harinarayan et al (2006); Kavoosi et al (2007); Langford et al (2007); Ljunglöf et al (2007); Susanto et al (2007).…”