title: miRNA profiling of high, low and non-producing CHO cells during biphasic fed-batch cultivation reveals process relevant targets for host cell engineering.
abstract: Fed-batch cultivation of recombinant Chinese hamster ovary (CHO) cell lines is one of the most widely used production modes for commercial manufacturing of recombinant protein therapeutics. Furthermore, fed-batch cultivations are often conducted as biphasic processes where the culture temperature is decreased to maximize volumetric product yields. However, it remains to be elucidated which intracellular regulatory elements actually control the observed pro-productive phenotypes. Recently, several studies have revealed microRNAs (miRNAs) to be important molecular switches of cell phenotypes. In this study, we analyzed miRNA profiles of two different recombinant CHO cell lines (high and low producer), and compared them to a non-producing CHO DG44 host cell line during fed-batch cultivation at 37°C versus a temperature shift to 30°C. Taking advantage of next-generation sequencing combined with cluster, correlation and differential expression analyses, we could identify 89 different miRNAs, which were differentially expressed in the different cell lines and cultivation phases. Functional validation experiments using 19 validated target miRNAs confirmed that these miRNAs indeed induced changes in process relevant phenotypes. Furthermore, computational miRNA target prediction combined with functional clustering identified putative target genes and cellular pathways, which might be regulated by these miRNAs. This study systematically identified novel target miRNAs during different phases and conditions of a biphasic fed-batch production process and functionally evaluated their potential for host cell engineering.
authors.affiliation: Institute of Applied Biotechnology, University of Applied Sciences Biberach, Hubertus-Liebrecht-Strasse 35, 88400 Biberach, Germany; University of Ulm, Faculty of Medicine, Albert-Einstein-Allee 11, 89079 Ulm, Germany. Electronic address: stiefel@hochschule-bc.de.
authors.forename: Fabian
authors.initials: F
authors.lastname: Stiefel
authors.affiliation: Institute of Applied Biotechnology, University of Applied Sciences Biberach, Hubertus-Liebrecht-Strasse 35, 88400 Biberach, Germany; University of Ulm, Faculty of Medicine, Albert-Einstein-Allee 11, 89079 Ulm, Germany. Electronic address: simon.fischer@boehringer-ingelheim.com.
authors.forename: Simon
authors.initials: S
authors.lastname: Fischer
authors.affiliation: University of Bielefeld, Center of Biotechnology (CeBiTec), 33501 Bielefeld, Germany. Electronic address: asczyrba@techfak.uni-bielefeld.de.
authors.forename: Alexander
authors.initials: A
authors.lastname: Sczyrba
authors.affiliation: Institute of Applied Biotechnology, University of Applied Sciences Biberach, Hubertus-Liebrecht-Strasse 35, 88400 Biberach, Germany. Electronic address: otte@hochschule-bc.de.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Otte
authors.affiliation: Institute of Applied Biotechnology, University of Applied Sciences Biberach, Hubertus-Liebrecht-Strasse 35, 88400 Biberach, Germany. Electronic address: hesse@hochschule-bc.de.
authors.forename: Friedemann
authors.initials: F
authors.lastname: Hesse
mesh_terms: D000818:Animals; D061249:Batch Cell Culture Techniques / Q000379:methods*; D016466:CHO Cells; D060846:Cell Engineering / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D020869:Gene Expression Profiling / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing; D035683:MicroRNAs / Q000235:genetics*; D010641:Phenotype; D017423:Sequence Analysis, RNA / Q000379:methods
chemical_list: D035683:MicroRNAs
keywords: CHO; Fed-Batch; Next generation sequencing; miRNA profiles