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Will Two-Phase Purification Replace Protein A?

Reconsider using aqueous two-phase systems (ATPS) in purification processes

 

Aqueous two-phase systems (ATPS) for protein separation and purification was a program theme at the Biopartitioning and Purification Conference 2015 (BPP 2015) held on June 7-10, 2015 in Vienna, Austria. This process, often involving polyethylene glycol, (PEG) and a phosphate, sulfate or citrate buffer for example, are based on partitioning a molecule between two phases. The rekindling of interest in ATPS has come about as the biopharmaceutical industry begins to look beyond Protein-A (PA) for monoclonal antibody (MAb) purification as titers continue to increase and immunotherapeutic molecules evolve.

For conventional MAbs processes, ATPS can effectively remove cells and other debris, reducing both the process volume and the burden on the clarification filters. As the harvest titer increases, the percentage of process impurities decreases, and the recovery and purity of the target molecule with ATPS increases. High titer, relatively large hydrophobic molecules (which include MAb), are ideal targets for a platform process utilizing ATPS and potentially crystallization. ATPS may also be a good choice for MAb or MAb-like molecules that are poorly suited to the Protein-A platform. For example, those antibodies that may dimerize or fragment at the low pH used to elute them from PA.

Although MAbs will continue to be an important part of the drug development pipeline for many years, there is a potential shift underway in the immunotherapy landscape to antibody-like molecules (e.g. FAb, Fab2, single chain antibodies, dimeric single chain antibodies, nanobodies, BiTES, etc.). The number of viral gene therapies, VLP’s and recombinant proteins emerging from discovery into development also appears to be increasing. These molecules are not amenable to a PA platform, but may be good candidates for ATPS, either for protein separation or for crystallography. Viruses and VLP are especially attractive candidates due to their large size and high partitioning coefficients.

Where can ATPS help? The BPP 2015 meeting discussed ATPS, crystallography, formulation and other subjects related to the understanding of protein separation, stability and solubilization. ATPS is especially useful for hydrophobic proteins such as IGF-1 and TPA and shows promise for the continuous refolding of GCSF. ATPS can improve protein solubility and protein separations, plus may modify both protein solubility and resin selectivity. Abraham Lenhoff, Professor of Chemical Engineering at the University of Delaware, reported that the insoluble material formed at high protein concentrations has structure. Using Newtonian light scattering, he observed that these colloidal gels have a bicontinuous structure. This finding has implications for the separation of many molecules that are loaded at high concentrations on resins and then eluted with a steep step gradient. The elution profile may represent a series of precipitations and solubilizations as the protein concentration changes on its transit through the resin. Dariusch Hekmat, Staff Scientist at the Technical University of Munich (TUM), described the crystallization from crude solutions of a MAb having a genetically modified backbone to enhance crystallization. Understanding the Constant sequences that need to be changed and evaluating if these are acceptable changes from an immunological viewpoint, will be important moving forward. In conclusion, it’s my opinion that, ATPS, and the polymers used for ATPS, should be (re)considered in the purification, precipitation, resolubilization and stabilization of recombinant proteins (including MAbs) viruses and VLPs.

 

Blog article by: Frank Riske