title: Nonspecific interactions of chromatin with immunoglobulin G and protein A, and their impact on purification performance.
abstract: Chromatin released from dead host cells during in vitro production of IgG monoclonal antibodies exists mostly in complex hetero-aggregates consisting of nucleosomal arrays (DNA+histone proteins), non-histone proteins, and aberrant forms of IgG. They bind immobilized protein A more aggressively than IgG, through their nucleosomal histone components, and hinder access of IgG to Fc-specific binding sites, thereby reducing dynamic binding capacity. The majority of host cell contaminants in eluted IgG are leachates from chromatin hetero-aggregates that remain bound to protein A. Formation of turbidity in eluted IgG during pH titration is caused by neutral-pH insolubility of chromatin hetero-aggregates. NaOH is required at 500 mM to remove accumulated chromatin. A chromatin-directed clarification method removed 99% of histones, 90% of non-histone proteins, achieved a 6 log reduction of DNA, 4 log reduction of lipid-enveloped virus, and 5 log reduction of non-enveloped retrovirus, while conserving 98% of the native IgG. This suspended most of performance compromises imposed on protein A. IgG binding capacity increased ~20%. Host protein contamination was reduced about 100-fold compared to protein A loaded with harvest clarified by centrifugation and microfiltration. Aggregates were reduced to less than 0.05%. Turbidity of eluted IgG upon pH neutralization was nearly eliminated. Column cleaning was facilitated by minimizing the accumulation of chromatin.
authors.affiliation: Bioprocessing Technology Institute, 20 Biopolis Way, Centros No. 06-01, Singapore 138668, Singapore. Electronic address: pete_gagnon@mac.com.
authors.forename: Pete
authors.initials: P
authors.lastname: Gagnon
authors.affiliation: Bioprocessing Technology Institute, 20 Biopolis Way, Centros No. 06-01, Singapore 138668, Singapore.
authors.forename: Rui
authors.initials: R
authors.lastname: Nian
authors.affiliation: Bioprocessing Technology Institute, 20 Biopolis Way, Centros No. 06-01, Singapore 138668, Singapore.
authors.forename: Jeremy
authors.initials: J
authors.lastname: Lee
authors.affiliation: Bioprocessing Technology Institute, 20 Biopolis Way, Centros No. 06-01, Singapore 138668, Singapore.
authors.forename: Lihan
authors.initials: L
authors.lastname: Tan
authors.affiliation: Bioprocessing Technology Institute, 20 Biopolis Way, Centros No. 06-01, Singapore 138668, Singapore.
authors.forename: Sarah Maria Abdul
authors.initials: SM
authors.lastname: Latiff
authors.affiliation: Bioprocessing Technology Institute, 20 Biopolis Way, Centros No. 06-01, Singapore 138668, Singapore.
authors.forename: Chiew Ling
authors.initials: CL
authors.lastname: Lim
authors.affiliation: Bioprocessing Technology Institute, 20 Biopolis Way, Centros No. 06-01, Singapore 138668, Singapore.
authors.forename: Cindy
authors.initials: C
authors.lastname: Chuah
authors.affiliation: Bioprocessing Technology Institute, 20 Biopolis Way, Centros No. 06-01, Singapore 138668, Singapore.
authors.forename: Xuezhi
authors.initials: X
authors.lastname: Bi
authors.affiliation: Bioprocessing Technology Institute, 20 Biopolis Way, Centros No. 06-01, Singapore 138668, Singapore.
authors.forename: Yuansheng
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Bioprocessing Technology Institute, 20 Biopolis Way, Centros No. 06-01, Singapore 138668, Singapore.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Bioprocessing Technology Institute, 20 Biopolis Way, Centros No. 06-01, Singapore 138668, Singapore.
authors.forename: Hui Theng
authors.initials: HT
authors.lastname: Gan
mesh_terms: D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism*; D002846:Chromatography, Affinity; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification; D006657:Histones / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000302:isolation & purification* / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification; D013205:Staphylococcal Protein A / Q000378:metabolism*
chemical_list: D002843:Chromatin; D006657:Histones; D007074:Immunoglobulin G; D011506:Proteins; D013205:Staphylococcal Protein A; D004247:DNA
keywords: Aggregates; Capacity; Chromatin; IgG purification; Protein A; Turbidity