title: Immunoglobulin G elution in protein A chromatography employing the method of chromatofocusing for reducing the co-elution of impurities.
abstract: Purification processes for monoclonal Immunoglobulin G (IgG) typically employ protein A chromatography as a capture step to remove most of the impurities. One major concern of the post-protein A chromatography processes is the co-elution of some of the host cell proteins (HCPs) with IgG in the capture step. In this work, a novel method for IgG elution in protein A chromatography that reduces the co-elution of HCPs is presented where a two-step pH gradient is self-formed inside a protein A chromatography column. The complexities involved in using an internally produced pH gradient in a protein A chromatography column employing adsorbed buffering species are discussed though equation-based modeling. Under the conditions employed, ELISA assays show a 60% reduction in the HCPs co-eluting with the IgG fraction when using the method as compared to conventional protein A elution without affecting the IgG yield. Evidence is also obtained which indicates that the amount of leached protein A present in free solution in the purified product is reduced by the new method. Biotechnol. Bioeng. 2017;114: 154-162. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Department of Chemical, Biochemical and Environmental Engineering, University of Maryland Baltimore County, Baltimore, Maryland, 21250.
authors.forename: Nuno D S
authors.initials: ND
authors.lastname: Pinto
authors.affiliation: Department of Chemical, Biochemical and Environmental Engineering, University of Maryland Baltimore County, Baltimore, Maryland, 21250.
authors.forename: Shaunak D
authors.initials: SD
authors.lastname: Uplekar
authors.affiliation: Department of Chemical, Biochemical and Environmental Engineering, University of Maryland Baltimore County, Baltimore, Maryland, 21250.
authors.forename: Antonio R
authors.initials: AR
authors.lastname: Moreira
authors.affiliation: Department of Chemical, Biochemical and Environmental Engineering, University of Maryland Baltimore County, Baltimore, Maryland, 21250.
authors.forename: Govind
authors.initials: G
authors.lastname: Rao
authors.affiliation: Department of Chemical, Biochemical and Environmental Engineering, University of Maryland Baltimore County, Baltimore, Maryland, 21250.
authors.forename: Douglas D
authors.initials: DD
authors.lastname: Frey
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D004340:Drug Contamination / Q000517:prevention & control; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D011506:Proteins / Q000737:chemistry; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D011506:Proteins; D013205:Staphylococcal Protein A
keywords: chromatofocusing; immunoglobulin G; monoclonal antibody; protein A chromatography; protein purification