Prediction of salt effects on protein phase behavior by HIC retention and thermal stability.
In the biopharmaceutical industry it is mandatory to know and ensure the correct protein phase state as a critical quality attribute in every process step. Unwanted protein precipitation or crystallization can lead to column, pipe or filter blocking. In formulation, the formation of aggregates can even be lethal when injected into the patient. The typical methodology to illustrate protein phase states is the generation of protein phase diagrams. Commonly, protein phase behavior is shown in dependence of protein and precipitant concentration. Despite using high-throughput methods for the generation of phase diagrams, the time necessary to reach equilibrium is the bottleneck. Faster methods to predict protein phase behavior are desirable. In this study, hydrophobic interaction chromatography retention times were correlated to crystal size and form. High-throughput thermal stability measurements (melting and aggregation temperatures), using an Optim(®)2 system, were successfully correlated to glucose isomerase stability. By using hydrophobic interaction chromatography and thermal stability determinations, glucose isomerase conformational and colloidal stability were successfully predicted for different salts in a specific pH range.
Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
Kai
K
Baumgartner
Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
Steffen
S
Großhans
Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
Juliane
J
Schütz
Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
Susanna
S
Suhm
Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany. Electronic address: juergen.hubbuch@kit.edu.
Jürgen
J
Hubbuch
D019747:Aldose-Ketose Isomerases / Q000737:chemistry; D000645:Ammonium Sulfate; D002845:Chromatography; D003102:Colloids / Q000737:chemistry; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D010316:Particle Size; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D012015:Reference Standards; D012492:Salts / Q000737:chemistry*; D012965:Sodium Chloride
D003102:Colloids; D011506:Proteins; D012492:Salts; D012965:Sodium Chloride; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase; D000645:Ammonium Sulfate
High-throughput screening; Hydrophobic interaction chromatography; Protein phase diagram; Thermal stability