Protein A affinity chromatography is well established for the capture of monoclonal antibodies (mAbs) from mammalian cell culture supernatants. The protein A ligand interacts primarily with the Fc region of full mAbs. However, native protein A also binds to the variable region of the heavy chain subgroups VH3. As the majority of therapeutic Fab fragments in development are constructed from VH3 heavy chains, protein A affinity media would bind a greater part of these Fab molecules. A range of Fab-specific affinity resins has also become available over time, featuring ligands that bind to different sites of Fab molecules (constant region of kappa or lambda light chain, variable region of kappa light chain).<br/>We have examined the protein A affinity resin ProSep<sup>®</sup> Ultra Plus (PUP), exhibiting high dynamic binding capacity for mAbs and high flow rate capability, for the capture of Fab molecules from two different feedstocks. The dynamic binding capacities of PUP for both Fab molecules were comparable to a protein L based affinity media. PUP provided the fastest mass transfer reflected in the steepest breakthrough curve. Fab fragments were obtained in high purity (&gt;90 %) and high yield (&gt;95 %). With regard to its binding selectivity, PUP could be a more universal Fab binder compared to the other affinity approaches. Moreover, PUP is a proven media for mAb production processes allowing high reusability and process flexibility.

Protein A affinity chromatography for efficient purification of recombinant antibody fragments

249th National Meeting (2015)

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