pH Gradient Elution for Improved Separation of Monoclonal Antibody Charge Variants

Ion exchange is a very useful technique to separate protein mixtures under mild conditions, most commonly by applying a shallow gradient of increasing salt concentration as eluent. pH gradient elution is used much less frequently, except in specialist applications such as chromatofocusing, and can require complex buffer systems. However, today’s modern quaternary HPLC systems are ideally suited to generating pH gradient elution profiles from conventional buffer salts. In this application note, we demonstrate the benefits of using pH gradient elution for separation of charge isoforms of monoclonal antibodies using Agilent Bio MAb columns on an Agilent 1260 Infinity Bio-inert Quaternary LC system with Agilent Buffer Advisor software.