title: Determination of protein phase diagrams by microbatch experiments: exploring the influence of precipitants and pH.
abstract: Knowledge of protein phase behavior is essential for downstream process design in the biopharmaceutical industry. Proteins can either be soluble, crystalline or precipitated. Additionally liquid-liquid phase separation, gelation and skin formation can occur. A method to generate phase diagrams in high throughput on an automated liquid handling station in microbatch scale was developed. For lysozyme from chicken egg white, human lysozyme, glucose oxidase and glucose isomerase phase diagrams were generated at four different pH values – pH 3, 5, 7 and 9. Sodium chloride, ammonium sulfate, polyethylene glycol 300 and polyethylene glycol 1000 were used as precipitants. Crystallizing conditions could be found for lysozyme from chicken egg white using sodium chloride, for human lysozyme using sodium chloride or ammonium sulfate and glucose isomerase using ammonium sulfate. PEG caused destabilization of human lysozyme and glucose oxidase solutions or a balance of stabilizing and destabilizing effects for glucose isomerase near the isoelectric point. This work presents a systematic generation and extensive study of phase diagrams of proteins. Thus, it adds to the general understanding of protein behavior in liquid formulation and presents a convenient methodology applicable to any protein solution.
authors.affiliation: Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology, Engler-Bunte-Ring 1, Karlsruhe 76131, Germany.
authors.forename: Kai
authors.initials: K
authors.lastname: Baumgartner
authors.affiliation: Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology, Engler-Bunte-Ring 1, Karlsruhe 76131, Germany.
authors.forename: Lara
authors.initials: L
authors.lastname: Galm
authors.affiliation: Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology, Engler-Bunte-Ring 1, Karlsruhe 76131, Germany.
authors.forename: Juliane
authors.initials: J
authors.lastname: Nötzold
authors.affiliation: Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology, Engler-Bunte-Ring 1, Karlsruhe 76131, Germany.
authors.forename: Heike
authors.initials: H
authors.lastname: Sigloch
authors.affiliation: Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology, Engler-Bunte-Ring 1, Karlsruhe 76131, Germany.
authors.forename: Josefine
authors.initials: J
authors.lastname: Morgenstern
authors.affiliation: Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology, Engler-Bunte-Ring 1, Karlsruhe 76131, Germany.
authors.forename: Kristina
authors.initials: K
authors.lastname: Schleining
authors.affiliation: Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology, Engler-Bunte-Ring 1, Karlsruhe 76131, Germany.
authors.forename: Susanna
authors.initials: S
authors.lastname: Suhm
authors.affiliation: Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology, Engler-Bunte-Ring 1, Karlsruhe 76131, Germany.
authors.forename: Stefan A
authors.initials: SA
authors.lastname: Oelmeier
authors.affiliation: Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology, Engler-Bunte-Ring 1, Karlsruhe 76131, Germany. Electronic address: juergen.hubbuch@kit.edu.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Hubbuch
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000737:chemistry*; D000645:Ammonium Sulfate / Q000737:chemistry*; D011232:Chemical Precipitation; D003460:Crystallization; D005949:Glucose Oxidase / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D009113:Muramidase / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry*; D012965:Sodium Chloride / Q000737:chemistry*; D012996:Solutions
chemical_list: D012996:Solutions; D011092:Polyethylene Glycols; D012965:Sodium Chloride; D005949:Glucose Oxidase; D009113:Muramidase; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase; D000645:Ammonium Sulfate
keywords: Automated imaging; High throughput; Microbatch crystallization; Protein phase behavior; Protein phase diagram