title: miRNA engineering of CHO cells facilitates production of difficult-to-express proteins and increases success in cell line development.
abstract: In recent years, coherent with growing biologics portfolios also the number of complex and thus difficult-to-express (DTE) therapeutic proteins has increased considerably. DTE proteins challenge bioprocess development and can include various therapeutic protein formats such as monoclonal antibodies (mAbs), multi-specific affinity scaffolds (e.g., bispecific antibodies), cytokines, or fusion proteins. Hence, the availability of robust and versatile Chinese hamster ovary (CHO) host cell factories is fundamental for high-yielding bioprocesses. MicroRNAs (miRNAs) have emerged as potent cell engineering tools to improve process performance of CHO manufacturing cell lines. However, there has not been any report demonstrating the impact of beneficial miRNAs on industrial cell line development (CLD) yet. To address this question, we established novel CHO host cells constitutively expressing a pro-productive miRNA: miR-557. Novel host cells were tested in two independent CLD campaigns using two different mAb candidates including a normal as well as a DTE antibody. Presence of miR-557 significantly enhanced each process step during CLD in a product independent manner. Stable expression of miR-557 increased the probability to identify high-producing cell clones. Furthermore, production cell lines derived from miR-557 expressing host cells exhibited significantly increased final product yields in fed-batch cultivation processes without compromising product quality. Strikingly, cells co-expressing miR-557 and a DTE antibody achieved a twofold increase in product titer compared to clones co-expressing a negative control miRNA. Thus, host cell engineering using miRNAs represents a promising tool to overcome limitations in industrial CLD especially with regard to DTE proteins. Biotechnol. Bioeng. 2017;114: 1495-1510. © 2017 Wiley Periodicals, Inc.
authors.affiliation: Early Stage Bioprocess Development, Boehringer Ingelheim GmbH & Co. KG, Birkendorfer Strasse 65, 88397, Biberach, Germany.|Cell Culture Development CMB, Boehringer Ingelheim GmbH & Co. KG, Biberach, Germany.
authors.forename: Simon
authors.identifier: 0000-0002-5188-7620
authors.initials: S
authors.lastname: Fischer
authors.affiliation: Early Stage Bioprocess Development, Boehringer Ingelheim GmbH & Co. KG, Birkendorfer Strasse 65, 88397, Biberach, Germany.
authors.forename: Kim F
authors.initials: KF
authors.lastname: Marquart
authors.affiliation: Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany.
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Pieper
authors.affiliation: Early Stage Bioprocess Development, Boehringer Ingelheim GmbH & Co. KG, Birkendorfer Strasse 65, 88397, Biberach, Germany.
authors.forename: Juergen
authors.initials: J
authors.lastname: Fieder
authors.affiliation: Early Stage Bioprocess Development, Boehringer Ingelheim GmbH & Co. KG, Birkendorfer Strasse 65, 88397, Biberach, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Gamer
authors.affiliation: Early Stage Bioprocess Development, Boehringer Ingelheim GmbH & Co. KG, Birkendorfer Strasse 65, 88397, Biberach, Germany.
authors.forename: Ingo
authors.initials: I
authors.lastname: Gorr
authors.affiliation: Cell Culture Development CMB, Boehringer Ingelheim GmbH & Co. KG, Biberach, Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Schulz
authors.affiliation: Cell Culture Development CMB, Boehringer Ingelheim GmbH & Co. KG, Biberach, Germany.
authors.forename: Harald
authors.initials: H
authors.lastname: Bradl
mesh_terms: D000818:Animals; D061249:Batch Cell Culture Techniques / Q000379:methods*; D016466:CHO Cells / Q000166:cytology / Q000502:physiology*; D049109:Cell Proliferation / Q000502:physiology; D003412:Cricetulus; D024861:Genetic Enhancement / Q000379:methods*; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: C000621174:MIRN557 microRNA, human; D035683:MicroRNAs; D011994:Recombinant Proteins
keywords: Chinese hamster ovary (CHO) cells; cell engineering; difficult-to-express proteins; miR-557; microRNA; monoclonal antibody