Developing non-chromatographic strategies for biopharmaceuticals purification

Publication in peer-review paper dos Santos, R, Carvalho AL, Roque ACA. Renaissance of protein crystallization and precipitation in biopharmaceuticals purification. Biotechnol. Adv. 35 (2017) 41-50, https://doi.org/10.1016/j.biotechadv.2016.11.0The biopharmaceutical market shows a high annual growth, and the major contributors for this success are antibody molecules. The purification of high added value biological molecules is an important step in the manufacturing process of any biopharmaceutical, being currently focused on packed-bed chromatography, which does not cope with the high titers and working volumes obtained upstream. A new trend in downstream processing explores Anything But Chromatography (ABC) methodologies, including precipitation and crystallization techniques, as well as magnetic fishing. This work aims at developing a new ABC method - magnetic precipitation - which combines crystallization and affinity magnetic separation in a single step providing simultaneous protein purification and concentration. Firstly, the potential of small synthetic affinity ligands for protein capture was shown with the development of an affinity chromatographic adsorbent for human serum albumin capture. Secondly, the potential of affinity magnetic fishing in protein capture from complex mixtures was shown with affinity-functionalized magnetic particles targeting hen egg white lysozyme and antibodies from complex media, such as human serum plasma. As a first step to further investigate the potential of combining affinity magnetic particles in other ABC processes, we studied the influence of these particles in protein crystal growth as nucleation agents. As a proof-of-concept, functionalized and non-functionalized magnetic particles were used as additives in protein crystallization. A rational design for magnetic particles functionalization was achieved, having magnetic particles functionalized with affinity-triggering molecules for the crystallization of the target proteins - hen egg white lysozyme, bovine trypsin and green fluorescent protein from Aequorea victoria. The presence of functionalized magnetic particles did not hamper crystal growth nor the resolution of the diffracting crystal. The new magnetic crystallization method can overcome some protein crystallization difficulties, but also has the potential to be integrated in protein purification methods involving crystallization/precipitation steps. Finally, crystallization and magnetic fishing methods were used in synergy for antibody purification. A screening of precipitation conditions in the presence of magnetic particles functionalized with an affinity ligand for antibodies were tested against human plasma and mammalian cells supernatant. Affinity driven magnetic precipitation enabled crude extract fractionation and antibody recovery in the elution fraction with high purity and recovery yield, up to 99% and 97%, respectively. This work shows the development of a new method using crystallization and magnetic particles - magnetic precipitation, with the potential implications to be implemented and integrated as a platform to improve protein crystal for X-ray diffraction or as a step in a protein’s purification pipeline