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

JUAN M. GUREVICH MESSINA; SILVANA L. GIUDICESSI; MARA C. MARTINEZ-CERON; NICOLS URTASUN; GUILLERMINA FORNO; LAURA MAURO; OSVALDO CASCONE; SILVIA A. CAMPER

Proceedings of the 35th European Peptide Symposium

European Peptide Society

Lugar: Dublin; Año: 2018; p. 127 - 129

IntroductionHuman Follicle Stimulating Hormone(hFSH) is clinically used for ovulation in women and spermatogenesis inductionin men, in assisted reproduction technologies. As FSH-based biopharmaceuticals are parenterally administered, their purity must be high. Current methods for hFSH purification include severalchromatographic steps to reach the required purity. However, these involve a decrease in the hFSH total yield and rises the cost of the process. Affinity chromatography (AC) consists in the specific adsorption of target biomolecules onto ligands immobilized on chromatographic supports. Short peptides have been described as useful ligands for AC because of their low cost, simple chemical synthesis and high stability compared to protein-based ligands. The aim of this work was to design an affinity chromatography matrix with an immobilized synthetic peptide for rhFSH purification. MethodsIn a previous work, Sohn et al. (3) examined the hFSH receptor interaction with the hormone, testing each amino acid of the exoloop 3 by Ala substitution. Taking into account 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. The peptide was acetylated to increase its stability against possible attack by proteases present in the crude FSH sample. A Cyswas incorporated at the C-termini to facilitate its subsequent immobilization to the chromatographic activated SulfoLink agarose resin. A sample of crude rhFSH was loaded to the peptide affinity column using as adsorption and elution buffers: 20 mM sodium phosphate, 0.5 mM Met, pH 5.6 and 7.2 respectively.Results and DiscussionThe affinity chromatography support with the peptide ligand immobilized was evaluated by loading crudesamples composed of host cell proteins from the Chinese hamster ovary (CHO) cell and rhFSH (Fig. 1). A nonreducing SDS-PAGE was done to check rhFSH purification(Fig. 2). The purity obtained after AC purificationwas 94 % and the yield was 41% (Table 1). The highly glycosylated isoforms, which have the highest in vivo potency, were recovered. The identity of the protein band obtained in the SDS-PAGE was checked by peptide mapping analysis using LC-MS/MS. The peptide affinity column was overloaded, and the dynamic capacity obtained was 54.6 mg rhFSH/mL chromatographic resin.After its purification, rhFSH quality was analyzed. The percentage of oxidized rhFSH was 3.4 % and the percentage of free subunits was 1.2 %, both within the range established by the European Pharmacopeia as also were the sialic acid content and the isoforms profile.