Tuesday, November 9, 2010: 4:00 PM
250 A Room (Salt Palace Convention Center)
Three deamidation-associated variants of an experimental monoclonal antibody (mAb) were separated by cation-exchange chromatography. The adsorption equilibrium and kinetics were measured for two commercial cation exchangers: Capto S and UNOsphere S. The former is a crosslinked agarose matrix with dextran grafted inside the pores, while the latter consists of macroporous rigid particles. Single-component isotherm results showed that binding capacity for Capto S is generally higher than for UNOsphere S but decreases more substantially with increasing pH and salt concentration. On the other hand, binding strength differs for each variant at elevated pH condition, and becomes much weaker for the more deamidated species. A reasonable description of the isotherms was obtained with the steric mass action (SMA) model. The results show that the effective charge is the same for all isoforms, while the affinity constant varies, suggesting that differences in the overall charge, rather than differences in the number of surface-interacting charged residue, determine the selectivity. Accordingly, binary and ternary adsorption equilibrium could be predicted using the SMA. Batch adsorption kinetics with single-component system was studied at various pH and salt concentrations. The apparent effective pore diffusivity for Capto S is much higher than for UNOsphere S at low ionic strengths, but decrease dramatically at higher salt concentrations. Conversely, the effective diffusivities for UNOsphere S are much less affected by salt concentration and are consistent with those obtained for non-binding conditions. Simultaneous adsorption on Capto S shows patterns for each isoform that are similar to those obtained in single-component systems but with smaller magnitude of the saturated capacity for the more deamidated isoforms. In contrast, simultaneous adsorption on UNOsphere S shows temporary overshoots of the more weakly bound isoforms above equilibrium resulting from competitive binding. The adsorption behavior on Capto S matrix can be predicted well by a multi-component solid diffusion model assuming SMA equilibrium isotherm, while in UNOsphere S matrix the adsorption behavior can be described by a multi-component pore diffusion model along with SMA equilibrium adsorption.