Protein G affinity chromatography is an underrated but very potent purification method for a broad range of species-independent and tag-less Fab-fragments.

Because of their superior properties for certain biological applications small antibody derivatives like fragment of antigen binding (Fab) have found widespread use in basic research and as therapeutics. However, generation of Fab-fragments is still a rather complex matter, reflected by the fact that a variety of methods and purification techniques are necessary for the production of all the different classes of Fab-fragments (kappa/lambda light chains, type of species). Here we demonstrate that Fab-fragments derived from six different antibodies of human or murine origin produced by transient expression in HEK cells can be purified in a single step to a high degree of purity by standard protein G affinity chromatography. This is most likely due to alternative contact sites for protein G located in the CH1 domain of the Fab heavy chain. Our data demonstrate that protein G affinity chromatography as for whole antibodies is a robust method for the purification of tag-less Fab-fragments independent of species, significantly simplifying the process of Fab-fragment purification.