title: Improved recombinant antibody production by CHO cells using a production enhancer DNA element with repeated transgene integration at a predetermined chromosomal site.
abstract: Chinese hamster ovary (CHO) cells are one of the most useful host cell lines for the production of biopharmaceutical proteins. Although a series of production processes have been refined to improve protein productivity and cost performance, establishing producer cells is still time-consuming and labor-intensive. Recombinase-mediated site-specific gene integration into a predetermined chromosomal locus may enable predictable protein expression, reducing the laborious process of cell screening. We previously developed an accumulative site-specific gene integration system (AGIS) using Cre recombinase and mutated loxP sites for transgene integration and amplification in the CHO cell genome. Epigenetic modifier elements such as insulators are effective DNA cis-regulatory elements for stabilizing transgene expression. Here, we attempted to enhance transgene expression in recombinant CHO cells generated by AGIS using a production enhancer DNA element (PE) derived from the CHO genome. The PE was introduced into an expression unit for a recombinant scFv-Fc antibody. The effect on scFv-Fc productivity of PE position and orientation within the transgene was evaluated, while keeping the background chromosomal structure constant. For the optimal PE arrangement, scFv-Fc productivity was enhanced 2.6-fold compared with an expression unit without a PE. The enhancing effect of the PE on transgene expression was also observed when two or three PE-flanked expression units were inserted as tandem repeats. These results indicate that AGIS using the PE-flanked expression unit is a promising approach for establishing producer cell lines for biopharmaceutical protein production.
authors.affiliation: Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Kawabe
authors.affiliation: Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
authors.forename: Takanori
authors.initials: T
authors.lastname: Inao
authors.affiliation: Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
authors.forename: Shodai
authors.initials: S
authors.lastname: Komatsu
authors.affiliation: Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
authors.forename: Guan
authors.initials: G
authors.lastname: Huang
authors.affiliation: Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Ito
authors.affiliation: Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Omasa
authors.affiliation: Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan; Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan. Electronic address: kamihira@chem-eng.kyushu-u.ac.jp.
authors.forename: Masamichi
authors.initials: M
authors.lastname: Kamihira
mesh_terms: D000818:Animals; D001287:Attachment Sites, Microbiological / Q000235:genetics; D016466:CHO Cells; D002875:Chromosomes / Q000235:genetics*; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics; D005784:Gene Amplification / Q000235:genetics; D018390:Gene Targeting; D016678:Genome / Q000235:genetics; D019426:Integrases / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000235:genetics*; D020080:Tandem Repeat Sequences / Q000235:genetics; D019076:Transgenes / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D004247:DNA; C045073:Cre recombinase; D019426:Integrases
keywords: Accumulative gene integration; Cre/loxP; Production enhancer DNA element; Recombinant antibody production; Recombinase-mediated cassette exchange