title: Influence of macromolecular precipitants on phase behavior of monoclonal antibodies.
abstract: For the successful application of protein crystallization as a downstream step, a profound knowledge of protein phase behavior in solutions is needed. Therefore, a systematic screening was conducted to analyze the influence of macromolecular precipitants in the form of polyethylene glycol (PEG). First, the influence of molecular weight and concentration of PEG at different pH-values were investigated and analyzed in three-dimensional (3-D) phase diagrams to find appropriate conditions in terms of a fast kinetic and crystal size for downstream processing. In comparison to the use of salts as precipitant, PEG was more suitable to obtain compact 3-D crystals over a broad range of conditions, whereby the molecular weight of PEG is, besides the pH-value, the most important parameter. Second, osmotic second virial coefficients as parameters for protein interactions are experimentally determined with static light scattering to gain a deep insight view in the phase behavior on a molecular basis. The PEG-protein solutions were analyzed as a pseudo-one-compartment system. As the precipitant is also a macromolecule, the new approach of analyzing cross-interactions between the protein and the macromolecule PEG in form of the osmotic second cross-virial coefficient (B23 ) was applied. Both parameters help to understand the protein phase behavior. However, a predictive description of protein phase behavior for systems consisting of monoclonal antibodies and PEG as precipitant is not possible, as kinetic phenomena and concentration dependencies were not taken into account.
authors.affiliation: Section IV: Biomolecular Separation Engineering, Inst. of Engineering in Life Sciences, Karlsruhe Inst. of Technology, Engler-Bunte-Ring 1, 76131, Karlsruhe, Germany; Roche Diagnostics GmbH, 68305, Mannheim, Germany.
authors.forename: Natalie
authors.initials: N
authors.lastname: Rakel
authors.forename: Lara
authors.initials: L
authors.lastname: Galm
authors.forename: Katharina Christin
authors.initials: KC
authors.lastname: Bauer
authors.forename: Juergen
authors.initials: J
authors.lastname: Hubbuch
mesh_terms: D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000737:chemistry*; D003460:Crystallization; D006863:Hydrogen-Ion Concentration; D008027:Light; D046911:Macromolecular Substances / Q000737:chemistry*; D008956:Models, Chemical; D011092:Polyethylene Glycols / Q000737:chemistry; D012542:Scattering, Radiation
chemical_list: D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D011092:Polyethylene Glycols
keywords: crystallization; monoclonal antibody; phase behavior; protein interactions; static light scattering