Single step recombinant human follicle stimulating hormone purification by peptide affinity chromatography

JUAN M. GUREVICH MESSINA; SILVANA L. GIUDICESSI; MARÍA C. MARTÍNEZ-CERON; NICOLAS URTASUN; GUILLERMINA FORNO; LAURA MAURO; OSVALDO CASCONE; SILVIA A. CAMPERI

Simposio; 35th European Peptide Symposium; 2018

European Peptide Society

Human Follicle Stimulating Hormone (hFSH) is used clinically for womenovulation and men spermatogenesis induction, in assisted reproductiontechnologies. As FSH-based biopharmaceuticals are parenterally administered, their purity must be high. Current methods for hFSH purificationinclude several chromatographic steps to reach the required purity. However, these involve a decrease of the hFSH total yield, thus rising the costof the process. Short peptides have been described as useful ligands forAC because of their low cost, simple chemical synthesis and high stability in comparison to protein-based ligands. In previous works, Ryu et al(1998) and Sohn et al. (2002) studied the hFSH receptor and examinedthe interaction of its exoloop 3 with the hormone, testing each amino acidof that exoloop by Ala substitutions. From those works, the mutant withhigher affinity: (580)KVPLITVSKAK(590) was selected to design a synthetic ligand for affinity chromatography (AC): Ac-KVPLTVSKAKVACNH2. The peptide was synthesized as amide and was acetylated to avoidits polymerization during the coupling to the chromatographic support andto improve its stability to degradation by exopeptidases. A Cys was incorporated at the C-termini to facilitate its subsequent immobilization to thechromatographic activated SulfoLink agarose resin. A sample of crude recombinant FSH (rhFSH) was loaded to the peptide affinity column using20 mM sodium phosphate, 0.5 mM Met, pH 5.6 and 7.2 as adsorption andelution buffers respectively. The column was overloaded, and the dynamiccapacity obtained was 54.6 mg rhFSH/mL chromatographic resin. The purity obtained after AC purification was 95 %. Purified rhFSH quality wasanalyzed: the percentage of oxidized rhFSH was 3.4 % and the percentageof free subunits was 1 %, both of them in the range established by the European Pharmacopeia, as also were the sialic acid content and the isoformsprofile. The method here designed allows obtaining a high quality rhFSHusing a low-cost affinity matrix based on a short peptide ligand.