title: Engineering of novel Staphylococcal Protein A ligands to enable milder elution pH and high dynamic binding capacity.
abstract: We describe novel Staphylococcal Protein A ligands that enable milder elution pH for use in affinity chromatography. The change in elution pH is the result of point mutations to the protein sequence. Two novel ligands are investigated in this study. The first, designated Z(H18S)4, represents a histidine to serine substitution single mutation. The second, designated Z(H18S, N28A)4, is a double mutant comprising histidine to serine and asparagine to alanine mutations. Both are compared against the unmutated sequence, designated Z4, which is currently utilized in a commercially available Protein A stationary phase for the purification of molecules containing Fc domains. The ligands are coupled to a chromatography support matrix and tested against a panel of antibodies and an Fc fusion protein for elution pH, dynamic binding capacity, step-wise elution, and capture from clarified culture media. Results demonstrate that the novel ligands result in milder elution pH, on average >0.5 pH units, when tested in a pH gradient. For step-wise elution at pH 4.0, the Z(H18S, N28A)4 ligand showed on average a greater than 30% increase in yield compared to Z4. Importantly, for the antibodies tested the mutations did not result in a decrease in dynamic binding capacity or other desirable attributes such as selectivity. A potential application of the novel ligands is shown with a pH sensitive molecule prone to aggregation under acidic conditions.
authors.affiliation: MedImmune, Purification Process Sciences, One MedImmune Way, Gaithersburg, MD 20878, USA.
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Pabst
authors.affiliation: GE Healthcare, Life Sciences R&D, Bjorkgatan 30, SE-751 84 Uppsala, Sweden.
authors.forename: Ronnie
authors.initials: R
authors.lastname: Palmgren
authors.affiliation: GE Healthcare, Life Sciences R&D, Bjorkgatan 30, SE-751 84 Uppsala, Sweden.
authors.forename: Annika
authors.initials: A
authors.lastname: Forss
authors.affiliation: GE Healthcare, Life Sciences R&D, Bjorkgatan 30, SE-751 84 Uppsala, Sweden.
authors.forename: Jelena
authors.initials: J
authors.lastname: Vasic
authors.affiliation: MedImmune, Purification Process Sciences, One MedImmune Way, Gaithersburg, MD 20878, USA.
authors.forename: Mariko
authors.initials: M
authors.lastname: Fonseca
authors.affiliation: MedImmune, Purification Process Sciences, One MedImmune Way, Gaithersburg, MD 20878, USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: Thompson
authors.affiliation: MedImmune, Purification Process Sciences, One MedImmune Way, Gaithersburg, MD 20878, USA.
authors.forename: William K
authors.initials: WK
authors.lastname: Wang
authors.affiliation: MedImmune, Purification Process Sciences, One MedImmune Way, Gaithersburg, MD 20878, USA.
authors.forename: Xiangyang
authors.initials: X
authors.lastname: Wang
authors.affiliation: MedImmune, Purification Process Sciences, One MedImmune Way, Gaithersburg, MD 20878, USA. Electronic address: hunterak@medimmune.com.
authors.forename: Alan K
authors.initials: AK
authors.lastname: Hunter
mesh_terms: D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D016466:CHO Cells; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003412:Cricetulus; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D011994:Recombinant Proteins / Q000302:isolation & purification; D013205:Staphylococcal Protein A / Q000737:chemistry*
chemical_list: D000906:Antibodies; D008024:Ligands; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A
keywords: Affinity chromatography; Antibody; Protein A; Z domain