Online citations, reference lists, and bibliographies.

Utilizing A Regulated Targeted Integration Cell Line Development Approach To Systematically Investigate What Makes An Antibody Difficult To Express.

Tomofumi Tadauchi, Cynthia Sze-Lok Lam, Laura L. Liu, Yizhou Zhou, Danming Tang, Salina Louie, Brad Snedecor, Shahram Misaghi
Published 2019 · Chemistry, Medicine
Cite This
Download PDF
Analyze on Scholarcy
Chinese hamster ovary (CHO) cells are conventionally used to generate therapeutic cell lines via random integration (RI), where desired transgenes are stably integrated into the genome. Targeted integration (TI) approaches, which involve integration of a transgene into a specific locus in the genome, are increasingly utilized for CHO cell line development (CLD) in recent years. None of these CLD approaches, however, are suitable for expression of toxic or difficult-to-express molecules, or for determining the underlying causes for poor expression of some molecules. Here we introduce a regulated target integration (RTI) system, where the desired transgene is integrated into a specific locus and transcribed under a regulated promoter. This system was used to determine the underlying causes of low protein expression for a difficult-to-express antibody (mAb-A). Interestingly, we observed that both antibody heavy chain (HC) and light chain (LC) subunits of mAb-A independently contributed to its low expression. Analysis of RTI cell lines also revealed that while mAb-A LC triggered accumulation of intracellular BiP, its HC displayed impaired degradation and clearance. RTI pools, generated by swapping the WT or point-mutant versions of difficult-to-express antibody HC and LC with that of an average antibody, were instrumental in understanding the contribution of HC and LC subunits to the overall antibody expression. The ability to selectively turn off the expression of a target transgene in an RTI system could help to directly link expression of a transgene to an observed adverse effect. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2772, 2019.
This paper references
CHO cell engineering to prevent polypeptide aggregation and improve therapeutic protein secretion.
Valérie Le Fourn (2014)
The Unfolded Protein Response and Cell Fate Control.
Claudio Hetz (2018)
High Level Expression of Tissue Inhibitor of Metalloproteinases in Chinese Hamster Ovary Cells Using Glutamine Synthetase Gene Amplification
Mark I. Cockett (1990)
Post-translational modifications in the context of therapeutic proteins
Gary Walsh (2006)
Strategies and Considerations for Improving Expression of "Difficult to Express" Proteins in CHO Cells.
C. O. Sammya Alves (2017)
It's time to regulate: coping with product-induced nongenetic clonal instability in CHO cell lines via regulated protein expression.
Shahram Misaghi (2014)
Interaction of BiP with newly synthesized immunoglobulin light chain molecules : cycles of sequential binding and release
Knittler Mr (1992)
Taming hyperactive hDNase I: Stable inducible expression of a hyperactive salt‐ and actin‐resistant variant of human deoxyribonuclease I in CHO cells
Cynthia Lam (2017)
Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas
David G. Bole (1986)
CHO cells in biotechnology for production of recombinant proteins: current state and further potential
Jee Yon Kim (2011)
Recombinase-mediated cassette exchange (RMCE) for monoclonal antibody expression in the commercially relevant CHOK1SV cell line.
Lin Zhang (2015)
Towards rational engineering of cells: Recombinant gene expression in defined chromosomal loci
Kristina Nehlsen (2011)
Immunoglobulin Free Light Chain Dimers in Human Diseases
Batia Kaplan (2011)
A novel Bxb1 integrase RMCE system for high fidelity site-specific integration of mAb expression cassette in CHO Cells.
Mara C. Inniss (2017)
Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus.
Randal J. Kaufman (1991)
Expression systems for therapeutic glycoprotein production.
Yves Durocher (2009)
Transcriptional activation by tetracyclines in mammalian cells.
Manfred Gossen (1995)
Therapeutic antibodies for autoimmunity and inflammation
Andrew C Chan (2010)
Fast identification of reliable hosts for targeted cell line development from a limited-genome screening using combined φC31 integrase and CRE-Lox technologies.
Yongping Crawford (2013)
Detecting low level sequence variants in recombinant monoclonal antibodies
Yi Yang (2010)
Investigation of the free heavy chain homodimers of a monoclonal antibody
Hyo (Helen) Chung (2018)
Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.
Manfred Gossen (1992)
Immunoglobulin heavy chain binding protein
Ingrid G. Haas (1983)
Chromatin as a eukaryotic template of genetic information.
Giacomo Cavalli (2002)
Repeat-induced gene silencing in mammals
David Garrick (1998)
Model-directed engineering of "difficult-to-express" monoclonal antibody production by Chinese hamster ovary cells.
Leon P. Pybus (2014)
Promoter methylation and transgene copy numbers predict unstable protein production in recombinant Chinese hamster ovary cell lines.
Andrea Osterlehner (2011)
Transfected DNA is mutated in monkey, mouse, and human cells.
Jane S. Lebkowski (1984)
Advances in Mammalian Cell Line Development Technologies for Recombinant Protein Production
Tingfeng Lai (2013)
Recombinant protein expression by targeting pre-selected chromosomal loci
Kristina Nehlsen (2009)

This paper is referenced by
Semantic Scholar Logo Some data provided by SemanticScholar