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

Adsorption equilibrium and kinetics of monomer–dimer monoclonal antibody mixtures on a cation exchange resin

“…For this purpose, monomer and purified dimer samples were fluorescently labeled with amine-reactive dyes Rhodamine Red TM -X and Green TM -X, respectively, by incubating 1 mL of solutions containing 8 mg/mL protein in 500 mM NaHCO 3 , pH 8.5 buffer with each reactive dye in darkness for 1 h following the method in Refs. [31,32]. The dye-to-protein molar ratio was 1:3 for the monomer and 1:6 for the dimer, which resulted in average molar labeling ratios of 0.24 and 0.16 for the monomer and dimer, respectively, determined spectrophotometrically based on the dye extinction coefficients at 571 and 503 nm for rhodamine red and rhodamine green protein conjugates, respectively.…”

“…However, while the rate was essentially independent of the direction of transport for the macroporous resin, the rates observed for the polymer-grafted resin were different dependent on whether native and deamidated isoforms were adsorbed simultaneously or in a consecutive manner, with the native species displacing the deamidated isoforms. Tao et al [31], Perez et al [32], and Zhu and Carta [33] also used CLSM to study the evolution of intraparticle concentration profiles during adsorption of protein mixtures in CEX and AEX resins for, respectively, deamidated mAb mixtures, multiple mAbs, and mixtures of native and PEGylated proteins. These authors considered both macroporous and polymer-grafted resins.…”

Protein Adsorption and Transport in Novel Chromatographic Anion Exchange and Multimodal Resins

“…The behavior of multicomponent adsorption under solid diffusion mechanism is also very different from that under pore diffusion mechanism. For pore diffusion, protein can move in either direction towards or away from the particle center while for solid diffusion in polymer grafted materials counterdiffusion is highly hindered, as reported by Tao et al [46] and Almodovar et al [23] for the adsorption of a mAb on polymer grafted cation exchangers.…”

“…In this case, protein molecules reach the center of the particle quickly but approach equilibrium slowly. This behavior has been reported by many researchers, mainly for the stationary phases that have small mesh pore size or have incorporated a grafted polymers or soft gel phase within a large porous structure [3,20,46].…”

“…For example, Tao et al observed smooth intraparticle protein concentration profiles for the adsorption of mAb variants in Capto S, a dextran grafted cation exchanger [46]. The behavior of multicomponent adsorption under solid diffusion mechanism is also very different from that under pore diffusion mechanism.…”