title: Biphasic cultivation strategy to avoid Epo-Fc aggregation and optimize protein expression.
abstract: In biphasic cultivations, the culture conditions are initially kept at an optimum for rapid cell growth and biomass accumulation. In the second phase, the culture is shifted to conditions ensuring maximum specific protein production and the protein quality required. The influence of specific culture parameters is cell line dependent and their impact on product quality needs to be investigated. In this study, a biphasic cultivation strategy for a Chinese hamster ovary (CHO) cell line expressing an erythropoietin fusion protein (Epo-Fc) was developed. Cultures were run in batch mode and after an initial growth phase, cultivation temperature and pH were shifted. Applying a DoE (Design of Experiments) approach, a fractional factorial design was used to systematically evaluate the influence of cultivation temperature and pH as well as their synergistic effect on cell growth as well as on recombinant protein production and aggregation. All three responses were influenced by the cultivation temperature. Additionally, an interaction between pH and temperature was found to be related to protein aggregation. Compared with the initial standard conditions of 37°C and pH 7.05, a parameter shift to low temperature and acidic pH resulted in a decrease in the aggregate fraction from 75% to less than 1%. Furthermore, the synergistic effect of temperature and pH substantially lowered the cell-specific rates of glucose and glutamine consumption as well as lactate and ammonium production. The optimized culture conditions also led to an increase of the cell-specific rates of recombinant Epo-Fc production, thus resulting in a more economic bioprocess.
authors.affiliation: BioMarin International Limited, Shanbally, Ringaskiddy, County Cork, Ireland. Electronic address: christian.kaisermayer@bmrn.com.
authors.forename: Christian
authors.initials: C
authors.lastname: Kaisermayer
authors.affiliation: Vienna Institute of BioTechnology, Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Muthgasse 11, 1190 Vienna, Austria. Electronic address: david.reinhart@boku.ac.at.
authors.forename: David
authors.initials: D
authors.lastname: Reinhart
authors.affiliation: Polymun Scientific Immunbiologische Forschung GmbH, Donaustraße 99, 3400 Klosterneuburg, Austria. Electronic address: andreas.gili@polymun.com.
authors.forename: Andreas
authors.initials: A
authors.lastname: Gili
authors.affiliation: Polymun Scientific Immunbiologische Forschung GmbH, Donaustraße 99, 3400 Klosterneuburg, Austria. Electronic address: martina.chang@polymun.com.
authors.forename: Martina
authors.initials: M
authors.lastname: Chang
authors.affiliation: GE Healthcare Life Sciences AB, Björkgatan 30, 75184 Uppsala, Sweden. Electronic address: per-mikael.aberg@ge.com.
authors.forename: Per-Mikael
authors.initials: PM
authors.lastname: Aberg
authors.affiliation: GE Healthcare Life Sciences AB, Björkgatan 30, 75184 Uppsala, Sweden. Electronic address: andreas.castan@ge.com.
authors.forename: Andreas
authors.initials: A
authors.lastname: Castan
authors.affiliation: Vienna Institute of BioTechnology, Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Muthgasse 11, 1190 Vienna, Austria. Electronic address: renate.kunert@boku.ac.at.
authors.forename: Renate
authors.initials: R
authors.lastname: Kunert
mesh_terms: D000818:Animals; D016466:CHO Cells; D018929:Cell Culture Techniques / Q000379:methods*; D002850:Chromatography, Gel; D006224:Cricetinae; D003412:Cricetulus; D004921:Erythropoietin / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D055442:Metabolome; D066329:Protein Aggregates; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013696:Temperature
chemical_list: D066329:Protein Aggregates; D011993:Recombinant Fusion Proteins; D004921:Erythropoietin
keywords: Biphasic cultivation for bioprocess development; Chinese hamster ovary cell; Design of experiments; Parameter shift; Protein aggregation; Recombinant Epo-Fc production