The eluent composition strongly affects the pH of the product pool in protein A chromatography

Eluting protein A columns used to capture IgG antibodies and Fc-fusion proteins with low-pH eluents is shown to result in complex pH transients which occur regardless of protein loading and are caused primarily by interactions of buffer species with the protein A ligand. Using polyclonal IgG and the resin MabSelect PrismA as models, the behavior of glycine, acetate, and citrate elution buffers with respect to the ensuing pH and IgG elution profiles are examined experimentally as a function their molarity, pH, and flow velocity as well as protein loading. With glycine-based eluents, a slow moving pH wave propagates through the column resulting in fronting IgG elution peaks and high IgG elution pool pH values. Increasing the eluent pH from 3.2 to 4 slows the pH wave leading to increasing peak fronting and IgG pool pH. Increasing the eluent flow velocity broadens the upsloping portion of the IgG elution peak as a result of mass transfer limitations without affecting its downsloping portion. Eluents containing acetate or citrate result in minimal delay of the pH wave and sharper IgG peaks, but also low IgG elution pool pH values. The eluent flow velocity has minimal effects in this case. A previous model for elution with glycine at pH 3.2 is extended to higher pH values and to eluents containing acetate or citrate based on experimentally determined IgG adsorption isotherms as a function of pH and buffer type. Consistent with the data, the model predicts IgG binding strengths that increase in the order citrate > acetate > glycine. Thus, while for glycine nearly full recovery is obtained even with a pH 4 eluent, lower eluent pH values (3.2-3.5) are needed for acetate and, especially, citrate to achieve high recoveries of the bound IgG. From a practical viewpoint, the data suggest how the eluent makeup can be chosen to obtain IgG elution pools that have a desired pH. With more dilute glycine eluents at relatively high pH, an IgG elution pool with pH > 7 can be obtained with essentially complete IgG recovery at IgG loadings up to 45 mg/mL. The model can be used to understand the experimental trends and guide eluent selection.