Purification of the human fibroblast growth factor 2 using novel animal-component free materials

•Avoiding animal-based materials in protein purification.•Replacement of animal-based ligand heparin in affinity chromatography.•Alternative ligands can be mixed-mode and pseudo-biospecific ligands.•Mixed-mode chromatography as an alternative to heparin affinity chromatography. This paper analyzes the use of animal-component free chromatographic materials for the efficient purification of the human fibroblast growth factor 2 (hFGF-2). hFGF-2 is produced in Escherichia coli and purified via three different chromatography steps, which include a strong cation exchange chromatography as a capture step, followed by heparin affinity chromatography and an anion exchange chromatography as a polishing step. The affinity chromatography step is based on the animal-derived material heparin. Chemically produced ligands provide a viable alternative to animal-derived components in production processes, since they are characterized by a defined structure which leads to reproducible results and a broad range of applications. The alternative ligands can be assigned to adsorber of the mixed-mode chromatography (MMC) and pseudo-affinity chromatography. Eight different animal-component free materials used as adsorbers in MMC or pseudo-affinity chromatography were tested as a substitute for heparin. The MMCs were cation exchangers characterized with further functional residues. The ligands of the pseudo-affinity chromatography were heparin-like ligands which are based on heparin's molecular structure. The alternative methods were tested as a capture step and in combination with another chromatographic step in the purification procedure of hFGF-2. In each downstream step purity, recovery and yield were analysed and compared to the conventional downstream process. Two types of MMC – the column ForesightTM NuviaTM cPrimeTM from Bio-Rad Laboratories and the column HiTrapTM CaptoTM MMC from GE Healthcare Life Sciences - can be regarded as effective animal-component free alternatives to the heparin - based adsorber.