title: Deep sequencing reveals different compositions of mRNA transcribed from the F8 gene in a panel of FVIII-producing CHO cell lines.
abstract: Coagulation factor VIII (FVIII) is one of the most complex biopharmaceuticals due to the large size, poor protein stability and extensive post-translational modifications. As a consequence, efficient production of FVIII in mammalian cells poses a major challenge, with typical yields two to three orders of magnitude lower than for antibodies. In the present study we investigated CHO DXB11 cells transfected with a plasmid encoding human coagulation factor VIII. Single cell clones were isolated from the pool of transfectants and a panel of 14 clones representing a dynamic range of FVIII productivities was selected for RNA sequencing analysis. The analysis showed distinct differences in F8 RNA composition between the clones. The exogenous F8-dhfr transcript was found to make up the most abundant transcript in the present clones. No correlation was seen between F8 mRNA levels and the measured FVIII productivity. It was found that three MTX resistant, nonproducing clones had different truncations of the F8 transcripts. We find that by using deep sequencing, in contrast to microarray technology, for determining the transcriptome from CHO transfectants, we are able to accurately deduce the mature mRNA composition of the transgene and identify significant truncations that would probably otherwise have remained undetected.
authors.affiliation: Mammalian Cell Technology, Novo Nordisk A/S, Maaloev, Denmark. csrk@novonordisk.com.|Department of Systems Biology, Technical University of Denmark, Kgs Lyngby, Denmark. csrk@novonordisk.com.
authors.forename: Christian S
authors.initials: CS
authors.lastname: Kaas
authors.affiliation: Mammalian Cell Technology, Novo Nordisk A/S, Maaloev, Denmark.
authors.forename: Gert
authors.initials: G
authors.lastname: Bolt
authors.affiliation: Mammalian Cell Technology, Novo Nordisk A/S, Maaloev, Denmark.
authors.forename: Jens J
authors.initials: JJ
authors.lastname: Hansen
authors.affiliation: Department of Systems Biology, Technical University of Denmark, Kgs Lyngby, Denmark.
authors.forename: Mikael R
authors.initials: MR
authors.lastname: Andersen
authors.affiliation: Mammalian Cell Technology, Novo Nordisk A/S, Maaloev, Denmark.|Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Claus
authors.initials: C
authors.lastname: Kristensen
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005169:Factor VIII / Q000096:biosynthesis / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics*; D059467:Transcriptome / Q000235:genetics
chemical_list: D012333:RNA, Messenger; D011994:Recombinant Proteins; C078147:F8 protein, human; D005169:Factor VIII
keywords: CHO cells; Cell culture; Coagulation factor VIII; Gene delivery; Next-generation sequencing