Sophia Hober, PhD, Professor, School of Biotechnology, KTH Royal Institute of Technology
Antibodies are widely used affinity molecules in many fields of biological science and their use in therapy is constantly growing. The most common tool used for purification of antibodies is Protein A affinity chromatography, a method that offers high productivity and pure protein product. However, when eluting the captured antibodies from the column, low pH is needed which can be deleterious for certain antibodies and Fc-fusion proteins. We have addressed this issue by protein engineering. Through a combination of selection and directed mutagenesis the behavior of an IgG-binding domain from Protein A was improved resulting in ZCa, that allows for considerably milder elution of the captured antibody, since efficient release can be achieved by merely adding sodium chloride. This results in the release of IgG1 at pH 6 and antibodies of subclasses IgG2 and IgG4 at neutral pH. In contrast to elution at low pH using a commercial Protein A resin, this mild elution has been shown to eliminate the formation of antibody aggregates in the capture step. A tetrameric version of ZCa has demonstrated a binding capacity comparable to commonly used Protein A resins, including exceptional selectivity and recovery. Further, the resin was proved to be highly robust by processing high antibody titers for a great number of cycles in a continuous antibody-production and purification process at pilot scale, rendering high yields. The mild and efficient purification strategy based on ZCa has the potential to enable the development of a broader range of antibodies, which cannot tolerate the current acidic conditions used in antibody capture.