title: Fast and scalable purification of a therapeutic full-length antibody based on process crystallization.
abstract: The potential of process crystallization for purification of a therapeutic monoclonal IgG1 antibody was studied. The purified antibody was crystallized in non-agitated micro-batch experiments for the first time. A direct crystallization from clarified CHO cell culture harvest was inhibited by high salt concentrations. The salt concentration of the harvest was reduced by a simple pretreatment step. The crystallization process from pretreated harvest was successfully transferred to stirred tanks and scaled-up from the mL-scale to the 1 L-scale for the first time. The crystallization yield after 24 h was 88-90%. A high purity of 98.5% was reached after a single recrystallization step. A 17-fold host cell protein reduction was achieved and DNA content was reduced below the detection limit. High biological activity of the therapeutic antibody was maintained during the crystallization, dissolving, and recrystallization steps. Crystallization was also performed with impure solutions from intermediate steps of a standard monoclonal antibody purification process. It was shown that process crystallization has a strong potential to replace Protein A chromatography. Fast dissolution of the crystals was possible. Furthermore, it was shown that crystallization can be used as a concentrating step and can replace several ultra-/diafiltration steps. Molecular modeling suggested that a negative electrostatic region with interspersed exposed hydrophobic residues on the Fv domain of this antibody is responsible for the high crystallization propensity. As a result, process crystallization, following the identification of highly crystallizable antibodies using molecular modeling tools, can be recognized as an efficient, scalable, fast, and inexpensive alternative to key steps of a standard purification process for therapeutic antibodies.
authors.affiliation: Institute of Biochemical Engineering, Technische Universität München, Boltzmannstrasse 15, Garching, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Smejkal
authors.forename: Neeraj J
authors.initials: NJ
authors.lastname: Agrawal
authors.forename: Bernhard
authors.initials: B
authors.lastname: Helk
authors.forename: Henk
authors.initials: H
authors.lastname: Schulz
authors.forename: Marion
authors.initials: M
authors.lastname: Giffard
authors.forename: Matthias
authors.initials: M
authors.lastname: Mechelke
authors.forename: Franziska
authors.initials: F
authors.lastname: Ortner
authors.forename: Philipp
authors.initials: P
authors.lastname: Heckmeier
authors.forename: Bernhardt L
authors.initials: BL
authors.lastname: Trout
authors.forename: Dariusch
authors.initials: D
authors.lastname: Hekmat
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D001709:Biotechnology / Q000379:methods*; D016466:CHO Cells; D018929:Cell Culture Techniques; D003198:Computer Simulation; D006224:Cricetinae; D003412:Cricetulus; D003460:Crystallization / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008958:Models, Molecular; D013696:Temperature; D014325:Tromethamine
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D014325:Tromethamine
keywords: CHO cell culture harvest; alternative to chromatography; purification process; scale-up; stirred process crystallization; therapeutic monoclonal antibody