title: Solvent modulation strategy for superior antibody monomer/aggregate separation in cation exchange chromatography.
abstract: Cation exchange chromatography (CEX) is an integral part of many downstream processes for monoclonal antibodies (mAbs). However, in some cases CEX methods with standard mobile phase conditions do not lead to a sufficient removal of soluble antibody aggregates. The addition of neutral polymers such as polyethylene glycol (PEG) to the mobile phase can improve the separation of proteins in IEC remarkably. The applicability of this solvent modulation technique is limited by protein precipitation at higher PEG concentrations. To overcome this limitation solubility enhancers like polyols and amino acids can be added to the mobile phase. These additives are known to inhibit PEG-induced protein precipitation in solution. This new solvent modulation strategy was tested with three different mAbs on two different CEX resins in the presence of PEG in combination with various solubility enhancers. In order to assess the general applicability of this method, mAbs were selected that show major differences with respect to their sensitivity to PEG-induced precipitation and monomer/aggregate resolution performance that is achieved by CEX under standard conditions. For all three mAbs precipitation could be prevented without elimination of the positive PEG-effect. The addition of solubility enhancers gives access to improved separation at elevated PEG concentrations and high protein loadings without running into precipitation issues. Our data indicate that this method is generically applicable and leads to a superior antibody monomer/aggregate separation.
authors.affiliation: Institute of Biochemistry, University of Applied Sciences Mannheim, Germany.
authors.forename: Simon
authors.initials: S
authors.lastname: Kluters
authors.affiliation: Institute of Biochemistry, University of Applied Sciences Mannheim, Germany. Electronic address: c.frech@hs-mannheim.de.
authors.forename: Christian
authors.initials: C
authors.lastname: Frech
authors.affiliation: Roche Pharmaceutical Research and Early Development, Large Molecule Research, Roche Innovation Center Penzberg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: von Hirschheydt
authors.affiliation: Roche Pharmaceutical Research and Early Development, Large Molecule Research, Roche Innovation Center Penzberg, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Schaubmar
authors.affiliation: F. Hoffmann-La Roche AG, Basel, Switzerland.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Neumann
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000302:isolation & purification*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D011092:Polyethylene Glycols / Q000737:chemistry; D012995:Solubility; D012997:Solvents / Q000737:chemistry*; D013012:Sorbitol / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D012997:Solvents; D011092:Polyethylene Glycols; D013012:Sorbitol
keywords: Aggregate removal; Mobile phase additives; Monoclonal antibodies (mAbs); Polyethylene glycol (PEG); Solubility enhancer; Solvent modulation