CAMPERI Silvia Andrea
congresos y reuniones científicas
Single step recombinant human follicle stimulating hormone purification by a short peptide affinity chromatography
J. M. GUREVICH MESSINA; S. L. SAAVEDRA; S. L. GIUDICESSI; M. C. MARTÍNEZ CERON; N. URTASUN; G. FORNO; L. MAURO; O. CASCONE; S. A. CAMPERI
Simposio; 35th European Peptide Symposium; 2018
Europen Peptide Society
Human Follicle stimulating hormone (hFSH) is clinically used in ovulation in woman and spermatogenesis induction in men in assisted reproduction technologies. As FSH-based biopharmaceuticals are parenterally administered, their purity must be high. Current methods for hFSH purification include several chromatographic steps to obtain the required purity. However, these involve a decrease of the hFSH total yield and rises the cost of the process. Short peptides have been described as useful ligands for AC because of their low cost, simple chemical synthesis and more stability in comparison to protein-based ligands. In previous works, Ryu et al (1998) and Sohn et al. (2002) studied the hFSH receptor and examined the interaction of its exoloop 3 with the hormone, testing each amino acid of that exoloop by Ala substitutions. In those works, the mutant with greater affinity: (580)KVPLITVSKAK(590) was selected to design a synthetic ligand for affinity chromatography (AC): Ac-KVPLTVSKAKVAC-NH2. The peptide was synthesized as amide and was acetylated to avoid its polymerization during its coupling to the chromatographic support. A Cys was incorporated at the C-termini to facilitate its subsequent attachment on the chromatographic activated SulfoLink resin. The peptide Ac-KVPLTVSKAKVAC-NH2 was immobilized in agarose. A sample of crude recombinant FSH (rhFSH) was loaded to the peptide affinity column using as adsorption and elution buffer 20 mM sodium phosphate, 0.5 mM Met, pH 5.6 and 7.2 respectively. The column was overloaded, and the dynamic capacity obtained was 54.6 mg rhFSH/mL chromatographic resin. The purity obtained after AC purification was 95 %. After its purification rhFSH quality was analyzed. The percentage of oxidized rhFSH was 3.4 % and the percentage of free subunits was 1.17 %, which were inside the range established by European Pharmacopeia as well as the sialic acid content and the isoforms profile. The method here designed allows obtaining a high quality rhFSH using a low cost affinity matrix bases in short peptide ligand.