Purification of Antibody Fragments via Hydrophobic Interactions

The importance of monoclonal antibodies (mAbs) as therapeutic agents is steadily increasing, and each year nearly 300 novel mAbs are being evaluated for their therapeutic effectivity. Interest has also been expressed in the use of antibody fragments as therapeutic agents. The physiochemical properties of the fragments differ significantly from that of intact antibodies, and the fragments can more easily be coupled to dyes, toxins and vesicles for diagnostic purposes, cancer therapy and improved drug delivery, respectively.

The purification of intact antibodies can be achieved using Protein A, which binds to the antibody Fc domain with high specificity and affinity. However, no such general ligand exists for the antigen-binding fragments. Here, a novel and robust platform for purification of antibody fragments is described based on interaction with detergent micellar aggregates. In the absence of specific ligands and chromatographic media, the purification of antigen binding F(ab’)2 fragments is achieved and uses clusters of non-ionic detergent micelles chelated via Fe2+ ions and a hydrophobic chelator.
The FabRICATOR® (IdeS) enzyme was used to digest a monoclonal antibody into the antigen-binding F(ab’)2 fragment and the crystallizable (Fc) fragment domain. Briefly, the theory behind the purification method is based on the theory that hydrophobicity strengthen the interaction between the detergent and the antibody. If either the Fc or the F(ab’)2 fragment is relatively more hydrophobic, this should in theory authorize their separation from each other.
The results presented in this article provide evidence for the use of the described purification method as a platform technology for purifying antigen-binding F(ab’)2 fragments, also in the presence of E.coli lysate. The effectiveness and use of the purification method for other antibody fragments (such as Fab fragments, single chain variable fragments and single domain antibodies) remain to be investigated.


Dhandapani et al., 2021. Purification of antibody fragments via interaction with detergent micellar aggregates. Scientific Reports. https://doi.org/10.1038/s41598-021-90966-1.

FabRICATOR – Below hinge digestion of IgG.