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

SILVANA L. GIUDICESSI; GUILLERMINA FORNO; S.A. CAMPERI; M.C. MARTINEZ-CERON; LAURA MAURO; GUREVICH MESSINA, JUAN MANUEL; URTASUN, NICOLÁS; O. CASCONE

Dublin

Congreso; 35 EPS European Peptide Symposium; 2018

Europen Peptide Society

Human Follicle Stimulating Hormone (hFSH) is used clinically for women ovulation and men spermatogenesis induction, 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 reach the required purity. However, these involve a decrease of the hFSH total yield, thus rising the cost of the process. Short peptides have been described as useful ligands for AC 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 examined the interaction of its exoloop 3 with the hormone, testing each amino acid of that exoloop by Ala substitutions. From those works, the mutant with higher 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 the coupling to the chromatographic support and to improve its stability to degradation by exopeptidases. A Cys was incorporated at the C-termini to facilitate its subsequent immobilization to the chromatographic activated SulfoLink agarose resin. A sample of crude recombinant FSH (rhFSH) was loaded to the peptide affinity column using 20 mM sodium phosphate, 0.5 mM Met, pH 5.6 and 7.2 as adsorption and elution buffers 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 %. Purified rhFSH quality was analyzed: the percentage of oxidized rhFSH was 3.4 % and the percentage of free subunits was 1 %, both of them in the range established by the European Pharmacopeia, as also were the sialic acid content and the isoforms profile. The method here designed allows obtaining a high quality rhFSH using a low-cost affinity matrix based on a short peptide ligand.