Biphasic cultivation strategy to avoid Epo-Fc aggregation and optimize protein expression

• Published in: Journal of biotechnology Vol. 227; pp. 3 - 9
• Main Authors: Kaisermayer, Christian, Reinhart, David, Gili, Andreas, Chang, Martina, Aberg, Per-Mikael, Castan, Andreas, Kunert, Renate
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 10.06.2016
• Subjects: Agglomeration; Animals; Biotechnology; Biphasic cultivation for bioprocess development; Cell Culture Techniques - methods; Chinese hamster ovary cell; CHO Cells; Chromatography, Gel; Cricetinae; Cricetulus; Cultivation; Culture; Design of experiments; Erythropoietin - metabolism; Hydrogen-Ion Concentration; Metabolome; Parameter shift; Protein Aggregates; Protein aggregation; Proteins; Recombinant; Recombinant Epo-Fc production; Recombinant Fusion Proteins - metabolism; Strategy; Temperature; Biphasic cultivation for bioprocess development; Protein aggregation; Chinese hamster ovary cell; Design of experiments; Parameter shift; Recombinant Epo-Fc production
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2016.03.054

•DoE was used to optimize a biphasic bioprocess for an aggregation-prone Epo-Fc fusion protein.•A synergistic effect of cultivation temperature and pH on product aggregation was found.•Aggregation was reduced from 75% to levels below 1%.•Optimized conditions lowered cell metabolism, nutrient consumption and by-product accumulation.•Optimized conditions increased cell-specific production rates resulting in a more economic bioprocess. 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.