Adsorption kinetics of deamidated antibody variants on macroporous and dextran-grafted cation exchangers. III. Microscopic studies

► Confocal microscopy provides fundamental insight about the kinetics and mechanism of adsorption of mAb charge variants in cation exchangers. ► Adsorbent architecture has profound influence on rates and mechanisms. ► Microscopic level understanding provides the basis for process modeling and quality by design in mAb capture and separation. The kinetics of single and multicomponent adsorption of deamidated monoclonal antibody (mAb) charge variants is investigated using confocal laser scanning microscopy for two commercial cation exchangers, one with an open macroporous structure – UNOsphere S – and the other with charged dextran grafts – Capto S. Markedly different intraparticle concentration profiles are obtained, being very sharp for UNOsphere S, indicating pore diffusion control, but much more diffuse for Capto S, consistent with a solid or surface diffusion mechanism. For single-component adsorption, the mAb effective pore diffusivities for UNOsphere S are approximately D e = 4.5 × 10 −8 and 8.3 × 10 −8 cm 2/s at pH 5 and 7.5, respectively, while effective solid diffusivities for Capto S are D s = 0.98 × 10 −9 and 5.0 × 10 −9 cm 2/s at pH 5 and 7.5, respectively. Two-component adsorption at pH 7.5, where the deamidated variants are bound selectively also showed markedly different profiles for the two matrices. UNOsphere S showed distinct adsorption zones within the particles indicating that multicomponent transport occurs with continuous displacement of the more deamidated variant by the less deamidated one. Capto S, however, showed no spatial resolution of the variants within the particle during co-adsorption and very slow mass transfer during sequential adsorption suggesting that protein counter-diffusion is severely hindered in this material.