Multicomponent adsorption of monoclonal antibodies on macroporous and polymer grafted cation exchangers.

We compare the rates of adsorption of two monoclonal antibodies (mAbs) with different adsorption properties on the cation exchangers UNOsphere™ S and Nuvia™ S. The former contains large open pores while the latter is based on a backbone matrix similar to UNOsphere™ S but also contains grafted charged polymers. Both single component and two-component adsorption are considered. Adsorption capacity and rates are much higher for Nuvia™ S indicating that protein interactions with the charged grafted polymers facilitate both binding and diffusional transport. Intraparticle concentration profiles obtained by confocal microscopy show sharp fronts for UNOsphere™ S but diffuse profiles for Nuvia™ S. Transport is thus controlled by pore diffusion for UNOsphere™ S but is described by a single file diffusion (SFD) mechanism for Nuvia™ S. As a result, single and two-component adsorption occur at similar rates for UNOsphere™ S independent of the direction for transport. For Nuvia™ S, however, transport is very fast for single or two-component co-adsorption but very slow when counter diffusion of the two mAbs takes place within the particles. The transport models developed in this work allow a prediction of separation performance for overloaded conditions typical of process scale applications.

Counterion effects on protein adsorption equilibrium and kinetics in polymer-grafted cation exchangers.

Protein binding equilibrium and mass transfer kinetics are studied for cation exchangers containing charged polymer grafts as well as for a macroporous matrix in pH 5 acetate buffers using sodium, tetra-n-butylammonium (TBAH), arginine, and calcium as counterions and a monoclonal antibody (mAb) as a model protein. Dynamic light scattering shows that there is no significant effect of the counterion type on the mAb aqueous diffusivity. The counterion type also does not affect substantially the structure of the polymer grafts, nor does it affect the stoichiometry of the protein ion exchange process. While no counterion effects are also observed on the protein mass transfer kinetics for the macroporous matrix, very large effects are seen for the polymer grafted matrices with protein adsorption rates increasing dramatically in the order Ca⁺⁺> Arg⁺> Na⁺> TBAH⁺. This order is the same order in which the relative protein binding strength decreases. Accordingly, the counterions leading to weaker protein binding also lead to faster protein diffusion. Although the quantitative aspects are different, the same trends hold for different proteins (lysozyme and lactoferrin) and for an agarose-based matrix also containing grafted polymers (Capto™ S). The underlying mechanism is qualitatively consistent with protein transport occurring through a hopping process driven by the adsorbed protein concentration within the apparently flexible network structure formed by the grafted polymers. From a practical viewpoint, the results show that improved protein adsorption kinetics in polymer-grafted cation exchanger and, hence, improved performance, can be obtained by selecting particular counterions.

Protein adsorption and transport in cation exchangers with a rigid backbone matrix with and without polymeric surface extenders.

We compare the properties and protein adsorption characteristics of two polymeric cation exchangers: UNOsphere S, which has an open macroporous architecture, and Nuvia S, which is based on a very similar backbone matrix but contains sulfonated polymeric surface extenders. A monoclonal IgG and lysozyme were used as model adsorbates. The characteristic pore sizes, determined by inverse size exclusion chromatography, were about 140 nm for UNOsphere S, and only about 10 nm for Nuvia S, indicating that the polymeric extenders occupy a substantial portion of the base matrix pores. Greater exclusion limits were found for Nuvia S in 1 M NaCl and for a similar matrix containing uncharged surface extenders, suggesting that the polymeric extenders collapse partially at high ionic strength or when they are uncharged. Large equilibrium binding capacities were obtained for Nuvia S, approaching 320 ± 10 mg/mL of particle volume for both proteins in comparison with the UNOsphere S values of 170 ± 10 and 120 ± 10 mg/mL for lysozyme and IgG, respectively. Much higher adsorption rates were also found for Nuvia S, and the rate was nearly independent of protein concentration in solution. Confocal laser scanning microscopy showed very sharp intraparticle protein concentration profiles for UNOsphere S, consistent with a pore diffusion mechanism but diffuse concentration profiles for Nuvia S, consistent with a solid diffusion mechanism. The improved capacity and transport afforded by the polymeric extenders provide substantial potential benefits for bioprocess applications without sacrificing the desirable flow properties of the backbone matrix.

IgG adsorption on a new protein A adsorbent based on macroporous hydrophilic polymers II. Pressure-flow curves and optimization for capture.

Pressure-flow curves are obtained for a new protein A adsorbent matrix based on macroporous hydrophilic polymer beads with average diameter of 57 microm and a narrow particle size distribution. Experimental data are obtained in a 1cm diameter laboratory column and in preparative scale columns with diameters of 20, 30, and 45 cm. The results are consistent with a model that assumes a linear relationship between bed compression and relative flow velocity. Surprisingly, the packing compressibility is essentially independent of column diameter for the preparative columns. As a result, after accounting for the variation in extraparticle porosity caused by compression, the column pressure drop is accurately predictable using the Carman-Kozeny equation. A model is also developed to predict productivity for IgG capture as a function of operating conditions based on dynamic binding capacity data presented in Part I of this work. For typical conditions, the model predicts maximum productivity at low residence times, between 1 and 1.5 min, when the dynamic binding capacity is at about 70-80% of the maximum. Combining the two models for column pressure and for dynamic binding capacity allows the design of preparative scale columns that maximize productivity while meeting specified pressure constraints.

IgG adsorption on a new protein A adsorbent based on macroporous hydrophilic polymers. I. Adsorption equilibrium and kinetics.

Experimental determination and modeling of IgG binding on a new protein A adsorbent based on a macroporous resin were performed. The new adsorbent consists of polymeric beads based on hydrophilic acrylamido and vinyl monomers with a pore structure optimized to allow favorable interactions of IgG with recombinant protein A coupled to the resin. The particles have average diameter of 57 microm and a narrow particle size distribution. The IgG adsorption equilibrium capacity is 46 mg/cm(3) and the effective pore diffusivity determined from pulse response experiments for non-binding conditions is 8.0 x 10(-8) cm(2)/s. The IgG adsorption kinetics can be described with the same effective diffusivity by taking into account a heterogeneous binding mechanism with fast binding sites, for which adsorption is completely diffusion controlled, and slow binding sites for which adsorption is controlled by the binding kinetics. As a result of this mechanism, the breakthrough curve exhibits a tailing behavior, which appears to be associated with the slow binding sites. A detailed rate model taking into account intraparticle diffusion and binding kinetics is developed and is found capable of predicting both batch adsorption and breakthrough behavior over an ample range of experimental conditions. The corresponding effective diffusivity is independent of protein concentration in solution over the range 0.2-2 mg/cm(3) and of protein binding as a result of the large pore size of the support matrix. Overall, the small particle size and low diffusional hindrance allow capture of IgG with short residence times while attaining substantial dynamic binding capacities.