title: A potential generic downstream process using Cibracon Blue resin at very high loading capacity produces a highly purified monoclonal antibody preparation from cell culture harvest.
abstract: The use of a dye-ligand chromatography for the purification of monoclonal antibody (MAb) from cell culture and other feed streams has been largely overlooked in large scale production. Cibracon Blue dye (CB), a polycyclic anionic ligand, interacts with protein through a specific interaction between the dye, acting as a mimic of NAD+ and NADP+, or through non-specific electrostatic, hydrophobic, and other forces. In this paper, a CB resin was used to effectively and efficiently separate an IgG4 MAb from host and process impurities following the capture of the MAb on a Protein-A (PA) column. The CB unit operation, challenged at </=180 g MAb/L of resin with the PA eluate, reduced BSA (1-2 log), host cell protein (HCP; 2-3 log), MAb oligomer (31-85%), fragment (from approximately 0.8% to <0.1%), and other undesired MAb species. Purity, as measured by non-reducing (NR) SDS-PAGE, was improved 33-85%, to 92-99.5% overall (>99% by reducing SDS-PAGE). A facile three column scalable production scheme, employing CB as the second column in the process was used to generate highly purified MAb from cell culture harvest derived from two media of very different compositions. Free CB dye was </=1 ng/mg in MAb preparations purified through the three column process and then concentrated and buffer exchanged into the appropriate buffer using tangential flow filtration (TFF).
authors.affiliation: Purification Development, Genzyme Corporation, Framingham, MA 01701, USA. frank.riske@genzyme.com
authors.forename: F
authors.initials: F
authors.lastname: Riske
authors.forename: M
authors.initials: M
authors.lastname: Smith
authors.forename: M
authors.initials: M
authors.lastname: Menon
authors.forename: S
authors.initials: S
authors.lastname: Goetschalck
authors.forename: I V
authors.initials: IV
authors.lastname: Goidsenhoven
authors.forename: A
authors.initials: A
authors.lastname: Krul
authors.forename: V
authors.initials: V
authors.lastname: Pimpaneau
authors.forename: I
authors.initials: I
authors.lastname: Renaers
authors.forename: N
authors.initials: N
authors.lastname: Van Tichelt
authors.forename: K
authors.initials: K
authors.lastname: Van Houdt
authors.forename: M
authors.initials: M
authors.lastname: Hayes
authors.forename: C
authors.initials: C
authors.lastname: Lawrence
authors.forename: R
authors.initials: R
authors.lastname: Bigelow
authors.forename: J
authors.initials: J
authors.lastname: Schroeder
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000302:isolation & purification*; D045744:Cell Line, Tumor; D017077:Culture Media, Conditioned / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D007074:Immunoglobulin G / Q000276:immunology; D012117:Resins, Synthetic / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D017077:Culture Media, Conditioned; D007074:Immunoglobulin G; D012117:Resins, Synthetic