title: Design of experiments reveals critical parameters for pilot-scale freeze-and-thaw processing of L-lactic dehydrogenase.
abstract: Freezing constitutes an important unit operation of biotechnological protein production. Effects of freeze-and-thaw (F/T) process parameters on stability and other quality attributes of the protein product are usually not well understood. Here a design of experiments (DoE) approach was used to characterize the F/T behavior of L-lactic dehydrogenase (LDH) in a 700-mL pilot-scale freeze container equipped with internal temperature and pH probes. In 24-hour experiments, target temperature between -10 and -38°C most strongly affected LDH stability whereby enzyme activity was retained best at the highest temperature of -10°C. Cooling profile and liquid fill volume also had significant effects on LDH stability and affected the protein aggregation significantly. Parameters of the thawing phase had a comparably small effect on LDH stability. Experiments in which the standard sodium phosphate buffer was exchanged by Tris-HCl and the non-ionic surfactant Tween 80 was added to the protein solution showed that pH shift during freezing and protein surface exposure were the main factors responsible for LDH instability at the lower freeze temperatures. Collectively, evidence is presented that supports the use of DoE-based systematic analysis at pilot scale in the identification of F/T process parameters critical for protein stability and in the development of suitable process control strategies.
authors.affiliation: Research Center Pharmaceutical Engineering GmbH, Graz, Austria.|Institute for Biotechnology and Biochemical Engineering, Graz University of Technology, Austria.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Roessl
authors.affiliation: Research Center Pharmaceutical Engineering GmbH, Graz, Austria.|Institute for Biotechnology and Biochemical Engineering, Graz University of Technology, Austria.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Humi
authors.affiliation: Research Center Pharmaceutical Engineering GmbH, Graz, Austria.|Institute for Biotechnology and Biochemical Engineering, Graz University of Technology, Austria.
authors.forename: Stefan
authors.initials: S
authors.lastname: Leitgeb
authors.affiliation: Institute for Biotechnology and Biochemical Engineering, Graz University of Technology, Austria. bernd.nidetzky@tugraz.at.
authors.forename: Bernd
authors.initials: B
authors.lastname: Nidetzky
mesh_terms: D000818:Animals; D004867:Equipment Design; D005615:Freezing; D006863:Hydrogen-Ion Concentration; D007770:L-Lactate Dehydrogenase / Q000737:chemistry*; D008956:Models, Chemical; D018482:Muscle, Skeletal / Q000201:enzymology; D010865:Pilot Projects; D066329:Protein Aggregates; D055550:Protein Stability; D011817:Rabbits; D012107:Research Design
chemical_list: D066329:Protein Aggregates; D007770:L-Lactate Dehydrogenase
keywords: Design of experiments; Freezing/thawing; Protein aggregation; Protein stability; Quality by design