title: Characterization of a laboratory-scale container for freezing protein solutions with detailed evaluation of a freezing process simulation.
abstract: A 300-mL stainless steel freeze container was constructed to enable QbD (Quality by Design)-compliant investigations and the optimization of freezing and thawing (F/T) processes of protein pharmaceuticals at moderate volumes. A characterization of the freezing performance was conducted with respect to freezing kinetics, temperature profiling, cryoconcentration, and stability of the frozen protein. Computational fluid dynamic (CFD) simulations of temperature and phase transition were established to facilitate process scaling and process analytics as well as customization of future freeze containers. Protein cryoconcentration was determined from ice-core samples using bovine serum albumin. Activity, aggregation, and structural perturbation were studied in frozen rabbit muscle l-lactic dehydrogenase (LDH) solution. CFD simulations provided good qualitative and quantitative agreement with highly resolved experimental measurements of temperature and phase transition, allowing also the estimation of spatial cryoconcentration patterns. LDH exhibited stability against freezing in the laboratory-scale system, suggesting a protective effect of cryoconcentration at certain conditions. The combination of the laboratory-scale freeze container with accurate CFD modeling will allow deeper investigations of F/T processes at advanced scale and thus represents an important step towards a better process understanding.
authors: Research Center Pharmaceutical Engineering, Graz, Austria; Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Graz, Austria.
authors: Ulrich
authors: U
authors: Roessl
authors: Dalibor
authors: D
authors: Jajcevic
authors: Stefan
authors: S
authors: Leitgeb
authors: Johannes G
authors: JG
authors: Khinast
authors: Bernd
authors: B
authors: Nidetzky
mesh_terms: D000465:Algorithms; D000818:Animals; D002417:Cattle; D002626:Chemistry, Pharmaceutical / Q000295:instrumentation*; D002850:Chromatography, Gel; D003460:Crystallization; D004345:Drug Industry / Q000295:instrumentation; D004355:Drug Stability; D004867:Equipment Design; D005612:Freeze Drying; D005615:Freezing; D007770:L-Lactate Dehydrogenase / Q000737:chemistry; D009391:Nephelometry and Turbidimetry; D011506:Proteins / Q000737:chemistry*; D011817:Rabbits; D012710:Serum Albumin, Bovine / Q000737:chemistry; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013817:Thermography
chemical_list: D011506:Proteins; D012996:Solutions; D012710:Serum Albumin, Bovine; D007770:L-Lactate Dehydrogenase
keywords: HPLC; computational fluid dynamics; enzymes; fluorescence spectroscopy; freezing/thawing; protein aggregation; protein folding; protein stability; thermal analysis