Separation of protein mixtures using pH-gradient cation-exchange chromatography

Historically, separation of a protein mixture after adsorption to a cation-exchange column is effected by alteration in ionic strength. An alternative separation method using pH induced gradient in the range of 4–7.5 was studied. A cation-exchange column with large particle beads containing excessive carboxyls was employed. A pH gradient across the column was generated by a step change at the column entrance using a non-retained buffer system. Consistency and accuracy of pH values in timed intervals were demonstrated in three different batches. In development of the application, we found a correlation coefficient of >0.9 between the elution pH values of six acidic proteins and their isoelectric points. One case study showed the resolution between a monoclonal antibody and non-retained protein species from a protein A column. Another case study showed the feasibility of separating polyethylene glycol conjugated protein from native protein.