Online citations, reference lists, and bibliographies.

A High Cell Density Transient Transfection System For Therapeutic Protein Expression Based On A CHO GS-knockout Cell Line: Process Development And Product Quality Assessment.

Yashas Rajendra, Maria D Hougland, Riazul Alam, Teresa A Morehead, Gavin C. Barnard
Published 2015 · Chemistry, Medicine
Cite This
Download PDF
Analyze on Scholarcy
Share
Transient gene expression (TGE) is a rapid method for the production of recombinant proteins in mammalian cells. While the volumetric productivity of TGE has improved significantly over the past decade, most methods involve extensive cell line engineering and plasmid vector optimization in addition to long fed batch cultures lasting up to 21 days. Our colleagues have recently reported the development of a CHO K1SV GS-KO host cell line. By creating a bi-allelic glutamine synthetase knock out of the original CHOK1SV host cell line, they were able to improve the efficiency of generating high producing stable CHO lines for drug product manufacturing. We developed a TGE method using the same CHO K1SV GS-KO host cell line without any further cell line engineering. We also refrained from performing plasmid vector engineering. Our objective was to setup a TGE process to mimic protein quality attributes obtained from stable CHO cell line. Polyethyleneimine (PEI)-mediated transfections were performed at high cell density (4 × 10(6) cells/mL) followed by immediate growth arrest at 32 °C for 7 days. Optimizing DNA and PEI concentrations proved to be important. Interestingly, found the direct transfection method (where DNA and PEI were added sequentially) to be superior to the more common indirect method (where DNA and PEI are first pre-complexed). Moreover, the addition of a single feed solution and a polar solvent (N,N dimethylacetamide) significantly increased product titers. The scalability of process from 2 mL to 2 L was demonstrated using multiple proteins and multiple expression volumes. Using this simple, short, 7-day TGE process, we were able to successfully produce 54 unique proteins in a fraction of the time that would have been required to produce the respective stable CHO cell lines. The list of 54 unique proteins includes mAbs, bispecific antibodies, and Fc-fusion proteins. Antibody titers of up to 350 mg/L were achieved with the simple 7-day process. Titers were increased to 1 g/L by extending the culture to 16 days. We also present two case studies comparing product quality of material generated by transient HEK293, transient CHO K1SV GS-KO, and stable CHO K1SV KO pool. Protein from transient CHO was more representative of stable CHO protein compared to protein produced from HEK293.
This paper references
10.1016/j.pep.2009.12.015
Transient expression of an IL-23R extracellular domain Fc fusion protein in CHO vs. HEK cells results in improved plasma exposure.
Ka Fai Suen (2010)
10.1002/btpr.1693
A CHO cell line engineered to express XBP1 and ERO1-Lα has increased levels of transient protein expression.
Katharine Cain (2013)
10.1016/j.copbio.2004.08.002
Production technologies for monoclonal antibodies and their fragments.
Dana C. Andersen (2004)
Transient expression of an IL23R extracellular domain Fc fusion protein in CHO vs
KF Suen (2010)
transfection condition optimization. Protein Expr Purif
AB Bos (2014)
Accepted Preprint This article is protected by copyright. All rights reserved
10.5075/EPFL-THESIS-5572
A Simple High Yielding Transient Gene Expression System for CHO Cells
Yashas Rajendra (2013)
10.1038/nbt1026
Production of recombinant protein therapeutics in cultivated mammalian cells
F. M. Wurm (2004)
10.1002/btpr.1809
A high-yielding CHO transient system: coexpression of genes encoding EBNA-1 and GS enhances transient protein expression.
Olalekan Daramola (2014)
transient protein expression
I Kadura (2007)
10.1007/s10529-006-9297-y
Recombinant protein production by large-scale transient gene expression in mammalian cells: state of the art and future perspectives
Lucia Baldi (2006)
Mammalian cells. Method Enzymol
A Kichler (2005)
10.1016/S0076-6879(09)63015-9
Recombinant protein production by transient gene transfer into Mammalian cells.
S. Montecinos Geisse (2009)
10.1016/j.ymeth.2011.04.002
A simplified polyethylenimine-mediated transfection process for large-scale and high-throughput applications.
Céline Raymond (2011)
10.1016/j.pep.2008.10.017
Reflections on more than 10 years of TGE approaches.
S. Montecinos Geisse (2009)
10.1016/j.biotechadv.2011.08.022
Mammalian cell protein expression for biopharmaceutical production.
Jianwei Zhu (2012)
10.1038/nbt0910-917
Biopharmaceutical benchmarks 2010
Gary Walsh (2010)
10.1016/j.ijpharm.2009.08.032
Junk DNA enhances pEI-based non-viral gene delivery.
Ethlinn V B van Gaal (2010)
10.1016/j.jbiotec.2013.08.021
Development of a highly-efficient CHO cell line generation system with engineered SV40E promoter.
Lianchun Fan (2013)
10.1016/j.tim.2007.09.004
Microtiter plates as mini-bioreactors: miniaturization of fermentation methods.
Wouter A. Duetz (2007)
10.1002/btpr.1763
Use of an anti-apoptotic CHO cell line for transient gene expression.
Nichole Macaraeg (2013)
10.1002/jgm.805
Dilution of reporter gene with stuffer DNA does not alter the transfection efficiency of polyethylenimines.
A Kichler (2005)
DNA in reducing coding DNA requirement for transient gene expression with CHO and HEK293E cells
C Raymond (2011)
10.1016/j.jbiotec.2014.03.027
Development of a semi-automated high throughput transient transfection system.
Aaron B Bos (2014)
10.1002/bit.24365
Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells.
Lianchun Fan (2012)
10.1002/bit.24494
Role of non-specific DNA in reducing coding DNA requirement for transient gene expression with CHO and HEK-293E cells.
Yashas Rajendra (2012)
10.1002/bit.24514
Transient recombinant protein expression in a human amniocyte cell line: the CAP-T® cell system.
Simon Fischer (2012)
10.1016/j.pep.2011.02.008
Design of Experiment in CHO and HEK transient transfection condition optimization.
Frederic Bollin (2011)
10.1002/biot.201300468
Enhanced transient recombinant protein production in CHO cells through the co-transfection of the product gene with Bcl-xL.
Matthew P Zustiak (2014)
10.1016/j.jbiotec.2012.06.038
Differences in the glycosylation of recombinant proteins expressed in HEK and CHO cells.
Amélie Croset (2012)
10.1385/MB:34:2:225
Large-Scale transfection of mammalian cells for the fast production of recombinant protein
Phuong L. Pham (2006)
10.1093/nar/gkn423
Rational vector design and multi-pathway modulation of HEK 293E cells yield recombinant antibody titers exceeding 1 g/l by transient transfection under serum-free conditions
Gaurav Backliwal (2008)
10.1016/S0958-1669(02)00300-2
Recombinant protein expression for therapeutic applications.
Dana C. Andersen (2002)
10.1002/bit.21596
High-density transfection with HEK-293 cells allows doubling of transient titers and removes need for a priori DNA complex formation with PEI.
Gaurav Backliwal (2008)
10.1016/j.jbiotec.2011.03.001
A simple high-yielding process for transient gene expression in CHO cells.
Yashas Rajendra (2011)



This paper is referenced by
10.1002/9783527811410.ch3
Transient Gene Expression‐Based Protein Production in Recombinant Mammalian Cells
Joohyoung Lee (2019)
10.1007/s10616-019-00346-x
The transient expression of CHIKV VLP in large stirred tank bioreactors
Peifeng Chen (2019)
10.1002/bit.25888
Comparison of three transposons for the generation of highly productive recombinant CHO cell pools and cell lines.
Sowmya Balasubramanian (2016)
10.1016/J.COCHE.2018.09.010
Beyond preclinical research: production of CHO-derived biotherapeutics for toxicology and early-phase trials by transient gene expression or stable pools
Matthew Stuible (2018)
10.1002/biot.201700748
Generation of High Expressing Chinese Hamster Ovary Cell Pools Using the Leap-In Transposon System.
Sowmya Balasubramanian (2018)
10.3390/PR7060329
Enhanced Production of Anti-PD1 Antibody in CHO Cells through Transient Co-Transfection with Anti-Apoptotic Gene Bcl-xL Combined with Rapamycin
Yunxia Li (2019)
10.1002/btpr.2152
Enhanced plasmid DNA utilization in transiently transfected CHO-DG44 cells in the presence of polar solvents.
Yashas Rajendra (2015)
10.1002/ELSC.201800174
High cell density transient transfection of CHO cells for TGF-β1 expression.
Abdalla A. El-Shereef (2019)
10.1016/j.jbiotec.2018.06.307
Optimization of a high-cell-density polyethylenimine transfection method for rapid protein production in CHO-EBNA1 cells.
Matthew Stuible (2018)
10.1002/9783527699124.CH13
Expression Systems for Recombinant Biopharmaceutical Production by Mammalian Cells in Culture
Adam J Brown (2017)
10.1007/s10529-015-1938-6
Transcriptional and post-transcriptional targeting for enhanced transient gene expression in CHO cells
Yashas Rajendra (2015)
10.1016/j.jim.2016.09.007
Rapid high-throughput cloning and stable expression of antibodies in HEK293 cells.
Jared L Spidel (2016)
10.1007/978-1-4939-8730-6_3
PEI-Mediated Transient Gene Expression in CHO Cells.
Yashas Rajendra (2018)
10.1002/btpr.2527
Polymer‐mediated flocculation of transient CHO cultures as a simple, high throughput method to facilitate antibody discovery
Matthew G Schmitt (2017)
10.1002/9783527811410.ch2
Cell Line Development for Therapeutic Protein Production
Soo Min Noh (2019)
10.1007/s00449-018-1898-z
Enhanced production of anti-PD1 antibody in CHO cells through transient co-transfection with anti-apoptotic genes Bcl-xL and Mcl-1
Xinyu Zhang (2018)
10.1080/07388551.2017.1419459
Advancements in mammalian cell transient gene expression (TGE) technology for accelerated production of biologics
Sonia Gutiérrez-Granados (2018)
10.1007/978-1-4939-8730-6_1
Transient Gene Expression in Suspension HEK293-EBNA1 Cells.
Denis L'Abbé (2018)
10.1016/j.pep.2017.03.018
A high density CHO-S transient transfection system: Comparison of ExpiCHO and Expi293.
Nina K. Jain (2017)
10.1002/btpr.2307
Generation of stable Chinese hamster ovary pools yielding antibody titers of up to 7.6 g/L using the piggyBac transposon system.
Yashas Rajendra (2016)
Anticorps anti-cd9 thérapeutique
Hergen Spits (2017)
10.1007/10_2016_55
New Mammalian Expression Systems.
Jie Zhu (2018)
10.1002/btpr.2414
Transient and stable CHO expression, purification and characterization of novel hetero‐dimeric bispecific IgG antibodies
Yashas Rajendra (2017)
10.1002/pro.3240
Computational design of a specific heavy chain/&kgr; light chain interface for expressing fully IgG bispecific antibodies
K. J. Froning (2017)
10.1002/btpr.2724
Transient CHO expression platform for robust antibody production and its enhanced N-glycan sialylation on therapeutic glycoproteins.
Xiaotian Zhong (2019)
10.1002/btpr.2831
Development of an alternating tangential flow (ATF) perfusion-based transient gene expression (TGE) bioprocess for universal influenza vaccine.
Jinsung Hong (2019)
10.1051/medsci/2019219
[Biomanufacturing of monoclonal antibodies].
Simon Joubert (2019)
10.1007/7355_2016_3
Protein Production in Eukaryotic Cells
Kripa Ram (2016)
10.1007/s00253-018-8977-6
Co-amplification of EBNA-1 and PyLT through dhfr-mediated gene amplification for improving foreign protein production in transient gene expression in CHO cells
Joo-Hyoung Lee (2018)
Semantic Scholar Logo Some data provided by SemanticScholar