title: Advance chromatin extraction improves capture performance of protein A affinity chromatography.
abstract: Practical effects of advance chromatin removal on performance of protein A affinity chromatography were evaluated using a caprylic acid-allantoin-based extraction method. Lacking this treatment, the practice of increasing loading residence time to increase capacity was shown to increase host protein contamination of the eluted IgG. Advance chromatin extraction suspended that compromise. Protein A ligand leakage from columns loaded with chromatin-extracted harvest was half the level observed on protein A columns loaded with non-extracted harvest. Columns loaded with chromatin-extracted harvest were cleaned more effectively by 50-100mM NaOH than columns loaded with non-extracted harvest that were cleaned with 250-500mM NaOH. Two protein A media with IgG capacities in excess of 50g/L were loaded with chromatin-extracted harvest, washed with 2.0M NaCl before elution, and the eluted IgG fraction titrated to pH 5.5 before microfiltration. Host protein contamination in the filtrate was reduced to <1ppm, DNA to <1ppb, protein A leakage to 0.5ppm, and aggregates to 1.0%. Caprylic acid and allantoin were both reduced below 5ppm. Step recovery of IgG was 99.4%. Addition of a single polishing step reduced residual protein A beneath the level of detection and aggregates to <0.1%. Overall process recovery including chromatin extraction was 90%.
authors.affiliation: Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, 138668, Singapore.
authors.forename: Rui
authors.initials: R
authors.lastname: Nian
authors.affiliation: Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, 138668, Singapore.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, 138668, Singapore.
authors.forename: Lihan
authors.initials: L
authors.lastname: Tan
authors.affiliation: Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, 138668, Singapore.
authors.forename: Jeremy
authors.initials: J
authors.lastname: Lee
authors.affiliation: Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, 138668, Singapore.
authors.forename: Xeuzhi
authors.initials: X
authors.lastname: Bi
authors.affiliation: Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, 138668, Singapore.
authors.forename: Yuansheng
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, 138668, Singapore.
authors.forename: Hui Theng
authors.initials: HT
authors.lastname: Gan
authors.affiliation: Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, 138668, Singapore. Electronic address: pete_gagnon@bti.a-star.edu.sg.
authors.forename: Pete
authors.initials: P
authors.lastname: Gagnon
mesh_terms: D002843:Chromatin / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D007074:Immunoglobulin G / Q000737:chemistry; D008024:Ligands; D059625:Liquid-Liquid Extraction; D012972:Sodium Hydroxide / Q000737:chemistry; D013205:Staphylococcal Protein A / Q000737:chemistry*
chemical_list: D002843:Chromatin; D007074:Immunoglobulin G; D008024:Ligands; D013205:Staphylococcal Protein A; D012972:Sodium Hydroxide
keywords: Chromatin extraction; Cleaning efficiency; IgG purification; Ligand leakage; Loading residence time; Protein A