title: Examining the freezing process of an intermediate bulk containing an industrially relevant protein.
abstract: Numerous biopharmaceuticals are produced in recombinant microorganisms in the controlled environment of a bioreactor, a process known as Upstream Process. To minimize product loss due to physico-chemical and enzymatic degradation, the Upstream Process should be directly followed by product purification, known as Downstream Process. However, the Downstream Process can be technologically complex and time-consuming which is why Upstream and Downstream Process usually have to be decoupled temporally and spatially. Consequently, the product obtained after the Upstream Process, known as intermediate bulk, has to be stored. In those circumstances, a freezing procedure is often performed to prevent product loss. However, the freezing process itself is inseparably linked to physico-chemical changes of the intermediate bulk which may in turn damage the product. The present study analysed the behaviour of a Tris-buffered intermediate bulk containing a biopharmaceutically relevant protein during a bottle freezing process. Major damaging mechanisms, like the spatiotemporal redistribution of ion concentrations and pH, and their influence on product stability were investigated. Summarizing, we show the complex events which happen in an intermediate bulk during freezing and explain the different causes for product loss.
authors.affiliation: Institut für Luft- und Kältetechnik gemeinnützige Gesellschaft mbH, Dresden, Germany.
authors.forename: Holger
authors.initials: H
authors.lastname: Reinsch
authors.affiliation: Vienna University of Technology, Institute of Chemical Engineering, Research Area Biochemical Engineering, Vienna, Austria.
authors.forename: Oliver
authors.initials: O
authors.lastname: Spadiut
authors.affiliation: Institut für Luft- und Kältetechnik gemeinnützige Gesellschaft mbH, Dresden, Germany. Electronic address: johannes.heidingsfelder@ilkdresden.de.
authors.forename: Johannes
authors.initials: J
authors.lastname: Heidingsfelder
authors.affiliation: Vienna University of Technology, Institute of Chemical Engineering, Research Area Biochemical Engineering, Vienna, Austria; Laboratory for Mechanistic and Physiological Methods for Improved Bioprocesses, Institute of Chemical Engineering, Vienna University of Technology, Getreidemarkt 9/166, A-1060 Vienna, Austria. Electronic address: christoph.herwig@tuwien.ac.at.
authors.forename: Christoph
authors.initials: C
authors.lastname: Herwig
mesh_terms: D019149:Bioreactors; D001709:Biotechnology; D004795:Enzyme Stability; D005615:Freezing; D006735:Horseradish Peroxidase / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology; D010843:Pichia / Q000201:enzymology; D011994:Recombinant Proteins / Q000302:isolation & purification*; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D006735:Horseradish Peroxidase
keywords: Biopharmaceutical processing; Intermediate bulk; Pharmaceutical freezing; Physico-chemical changes; Product loss; pH shift