Recombinant human follicle stimulating hormone purification by a short peptide affinity chromatography

Abstract

Peptide KVPLITVSKAK was selected to design a synthetic ligand for affinity chromatography purification of recombinant human follicle stimulating hormone (rhFSH), based on the interaction of the hormone with the exoloop 3 of its receptor. The peptide was acetylated to improve its stability to degradation by exopeptidases. A cysteine was incorporated at the C‐termini to facilitate its immobilization to the chromatographic activated SulfoLink agarose resin. A sample of crude rhFSH was loaded to the affinity column, using 20 mM sodium phosphate, 0.5 mM methionine, and pH 5.6 and 7.2 as adsorption and elution buffers, respectively. The dynamic capacity of the matrix was 54.6 mg rhFSH/mL matrix and the purity 94%. The percentage of oxidized rhFSH was 3.4%, and that of the free subunits was 1.2%, both in the range established by the European Pharmacopeia, as also were the sialic acid content and the isoforms profile.

Peptide Affinity Chromatography Applied to Therapeutic Antibodies Purification

“…Thereby, immobilized peptide evaluation is preferred. A solid-phase strategy using 4-(hydroxymethyl)benzamide-ChemMatrix (HMBA-CM) resin and mass spectrometry (MS) has been developed to test peptide stability and applied in the selection of ligands for rhEPO, rhFSH, rhGH and bevacizumab purification by AC (Giudicessi et al 2017 ; Saavedra et al 2018 ; Gurevich Messina et al 2018 ; Barredo et al 2019 ). The method entitles: (a) solid-phase peptide synthesis on HMBA-CM resin; (b) peptidyl-resin beads incubation in solutions containing proteases or in cell culture broth; (c) whole peptide or C-terminal degradation products detachment from solid support with ammonia vapor; (d) MS sequencing of the peptide and C-terminal fragments (Fig.…”

“…Thereby, immobilized peptide evaluation is preferred. A solid-phase strategy using 4-(hydroxymethyl)benzamide-ChemMatrix (HMBA-CM) resin and mass spectrometry (MS) has been developed to test peptide stability and applied in the selection of ligands for rhEPO, rhFSH, rhGH and bevacizumab purification by AC (Giudicessi et al 2017;Saavedra et al 2018;Gurevich Messina et al 2018;Barredo et al 2019) (c) SPR affinity analysis: (c 1 ) peptide is separated from the solid support with ammonia vapor. (c 2 ) interactions between peptides and antibodies can be studied in real time by SPR without labeling the analytes of the peptide and C-terminal fragments (Fig.…”