Proteins biologics often contain several isoforms or variants, arising due to the differences in expression, post-translational modifications, protease action or chemical modification. These isoforms are best understood by evaluating their biological activity using bioassays that measure their biochemical or biological function. Availability of preparative amounts of these variants is essential for these assays. Many of the analytical techniques that are capable of separating these isoforms either do not provide material that can used readily or are not amenable to fractionation or scale-up.

In this study we report successful separation and/or enrichment of protein isoforms that arise due to differences in aggregation and sialation using preparative, high resolution pH based separation methods. Shallow gradients of over 20 column volumes over one pH unit provide a high resolution power, even under relatively high loads (2-5 mg/ml resin). Representative data from a couple of representative protein molecules that have been successfully separated using these techniques is presented.