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 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

miR‐CATCH Identifies Biologically Active miRNA Regulators of the Pro‐Survival Gene XIAP, in Chinese Hamster Ovary Cells

“…To navigate around this, microRNA‐based engineering has an added advantage owing to the non‐coding nature of these regulatory RNAs so as to not exert a translational burden on the CHO producer cell . Stable miRNA interference in a variety of CHO cell lines has generated improved phenotypes including higher product yields such as in the case of miR‐466h, miR‐2861, and miR‐557 . Given the limitations in miRNA target prediction, it is critical to identify bona fide miRNA regulators for the species, cell type, and gene under investigation as a means to increase the potential for a positive outcome.…”

“…Druz and colleagues reported the induction of apoptosis resistance in a CHO cell line stably engineered to be depleted of miR‐466h‐5p with an associated increase in the levels of anti‐apoptotic genes such as birc6 and bcl2l2 . Other studies across various research groups have further supported the utility of miRNA as cell engineering candidates including miR‐23, miR‐2861, miR‐17, and miR‐557 . In these instances, single miRNA engineering mediated a beneficial CHO cell phenotype via a multitude of molecular networks under regulation.…”

Integration of high‐throughput analytics and cell imaging enables direct early productivity and product quality assessment during Chinese Hamster ovary cell line development for a complex multi‐subunit vaccine antigen

“…Generation of stable cell lines which support high quantity and quality of target protein production is one of the first and most critical steps of process development for biologics. Conventional mammalian cell line development workflow includes: (a) stable pool generation; (b) single cell cloning with documented monoclonality to meet regulatory requirements; (c) clone screening based on protein yield and quality and (d) cell line bioprocess suitability testing (e.g., expression and genetic stability, and fed‐batch bioreactor process compatibility and scalability) …”

“…(b) single cell cloning with documented monoclonality to meet regulatory requirements; (c) clone screening based on protein yield and quality and (d) cell line bioprocess suitability testing (e.g., expression and genetic stability, and fed-batch bioreactor process compatibility and scalability). 1,2 The early clone screening stages are typically quite challenging.…”

Stable miRNA overexpression in human CAP cells: Engineering alternative production systems for advanced manufacturing of biologics using miR‐136 and miR‐3074

“…In addition, a miRNA screen was performed in HEK293 cells expressing a membrane protein fused to GFP (Xiao, Chen, Betenbaugh, Martin, & Shiloach, ). As an outcome, miRNAs now represent a powerful tool in cell line engineering (Barron, Sanchez et al, ; Jadhav et al, ) with proof of principle studies conducted in biopharmaceutical relevant CHO production cell lines (Fischer et al, ; Pieper, Strotbek et al, ; Pieper, Strotbek, Wenger, Olayioye, & Hausser, ). However, significant data are lacking for the novel CAP cells, although miRNA screens were performed in other human production cell lines (Xiao et al, ).…”