title: Performance of hexamer peptide ligands for affinity purification of immunoglobulin G from commercial cell culture media.
abstract: Previous work has reported on the identification and characterization of the hexapeptide ligands HWRGWV, HYFKFD, and HFRRHL for the affinity capture of IgG through specific binding to its Fc fragment. This paper addresses issues related to the successful application of these ligands, on a commercial methacrylate chromatographic resin, for the purification of IgG from mammalian cell culture fluids. The concentrations of sodium chloride and sodium caprylate in the binding buffer were optimized to maximize the purity and yield of IgG upon elution. Screening of several regeneration conditions found that either 2M guanidine-HCl or a combination of 0.85% phosphoric acid followed by 2M urea resulted in complete recovery of the IgG adsorption capacity and that the column could be reused over many cycles. The hexapeptide ligands were used for the purification of humanized and chimeric monoclonal antibodies from two commercial CHO cell culture fluids. The chimeric MAb of IgG1 subclass was purified using the HWRGWV resin whereas the humanized MAb of IgG4 subclass was purified using the HWRGWV, HYFKFD and HFRRHL resins. The purities and yields obtained for both the MAbs were found to be higher than 94% and 85% respectively. These results compare well with the yields and purities obtained using Protein G columns. The residual DNA and host cell protein reduction obtained by the HWRGWV resin was in the range of 4 log reduction value (LRV) and 2 LRV respectively, comparable to those reported for Protein A resins. The dynamic binding capacity of all three peptide resins for the humanized monoclonal antibody was in the range of 20mg/mL.
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, 1017 Main Campus Drive, Centennial Campus, Raleigh, NC 27695-7006, USA.
authors.forename: Amith D
authors.initials: AD
authors.lastname: Naik
authors.forename: Stefano
authors.initials: S
authors.lastname: Menegatti
authors.forename: Patrick V
authors.initials: PV
authors.lastname: Gurgel
authors.forename: Ruben G
authors.initials: RG
authors.lastname: Carbonell
mesh_terms: D000327:Adsorption; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000302:isolation & purification*; D016466:CHO Cells; D002210:Caprylates; D018929:Cell Culture Techniques; D002846:Chromatography, Affinity / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000302:isolation & purification*; D009842:Oligopeptides / Q000737:chemistry*; D010756:Phosphoric Acids; D011485:Protein Binding; D012965:Sodium Chloride; D014508:Urea
chemical_list: D000911:Antibodies, Monoclonal; D002210:Caprylates; D003470:Culture Media; D007074:Immunoglobulin G; D009842:Oligopeptides; D010756:Phosphoric Acids; D012965:Sodium Chloride; D014508:Urea; C030242:phosphoric acid; C031492:octanoic acid