Coexpression of acidic fibroblast growth factor enhances specific productivity and antibody titers in transiently transfected HEK293 cells

Backliwal, Gaurav; Hildinger, Markus; Chenuet, Sébastien; DeJesus, Maria; Wurm, Florian Μ.

doi:10.1016/j.nbt.2008.08.007

Cited by 21 publications

(12 citation statements)

References 14 publications

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“…Alternatively, considering cells were transfected with a small amount (2.5% (w/w)) of an XBP‐1 expressing plasmid that was both replication and retention deficient, dilution of XBP‐1 expression at an earlier stage of the process may also explain why no improvements in titer were observed. While such approaches to improving TGE remain in their infancy, these experiments further demonstrate the potential of co‐transfecting cells with transcription or growth factors to enhance recombinant protein yields from TGE 65…”

Section: Resultsmentioning

confidence: 93%

Efficient mAb production in CHO cells incorporating PEI‐mediated transfection, mild hypothermia and the co‐expression of XBP‐1

Codamo

Hou

Hughes

et al. 2011

J of Chemical Tech & Biotech

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BACKGROUND: Transient gene expression (TGE) provides a rapid way to generate recombinant protein biologics for pre-clinical assessment. Human embryonic kidney (HEK293) cells have traditionally been used for TGE; however, there is demand from industry for efficient, high-producing TGE systems that utilize Chinese hamster ovary (CHO) cells. A polyethyleneimine (PEI) -based TGE process has been developed for CHO cells using an episomal expression system to generate enhanced recombinant protein titers.

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Section: Resultsmentioning

confidence: 93%

Efficient mAb production in CHO cells incorporating PEI‐mediated transfection, mild hypothermia and the co‐expression of XBP‐1

Codamo

Hou

Hughes

et al. 2011

J of Chemical Tech & Biotech

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“…The high expression yields reported here were obtained with limited process optimization and development of the EBNA‐1‐GS host cell line. We anticipate additional significant increases in TGE yields by combining further process developments, such as supplementation of the transcriptional enhancer valproic acid with further cell engineering, such as coexpression with acidic fibroblast growth factor or through utilization of information obtained from the recently published CHO genome . A high‐yielding CHO TGE process presents the potential scenario of supplying clinical‐grade biopharmaceutical protein through a CHO transient process.…”

Section: Resultsmentioning

confidence: 99%

A high‐yielding CHO transient system: Coexpression of genes encoding EBNA‐1 and GS enhances transient protein expression

Daramola¹,

Stevenson²,

Dean³

et al. 2013

Biotechnology Progress

124

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An efficient rapid protein expression system is crucial to support early drug development. Transient gene expression is an effective route, and to facilitate the use of the same host cells as for subsequent stable cell line development, we have created a high-yielding Chinese hamster ovary (CHO) transient expression system. Suspension-adapted CHO-K1 host cells were engineered to express the gene encoding Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA-1) with and without the coexpression of the gene for glutamine synthetase (GS). Analysis of the transfectants indicated that coexpression of EBNA-1 and GS enhanced transient expression of a recombinant antibody from a plasmid carrying an OriP DNA element compared to EBNA-1-only transfectants. This was confirmed with the retransfection of an EBNA-1-only cell line with a GS gene. The retransfected cell lines showed an increase in transient expression when compared with that of the EBNA-1-only parent. The transient expression process for the best CHO transient cell line was further developed to enhance protein expression and improve scalability by optimizing the transfection conditions and the cell culture process. This resulted in a scalable CHO transient expression system that is capable of expressing 2 g/L of recombinant proteins such as antibodies. This system can now rapidly provide gram amounts of recombinant antibody to supply preclinical development studies that has comparable product quality to antibody produced from a stably transfected CHO cell line.

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“…Other enhancers are sodium butyrate (Leisy et al 2003;Mahonen et al 2007;Ping et al 2006) and trichostatin A (Spenger et al 2004). More recently proto-oncogenes, cell cycle control genes, growth factor genes and anti-apoptotic genes have been used to improve production hosts or transient expression (Wurm 2004;Backliwal et al 2008). Finally alternative cultivation methods for suspension cells are applied such as orbital shaker technology working under agitation within an incubator (Muller et al 2005) which allow cell densities up to 5 9 10 6 cells mL -1 while our cells reached densities of 1-2 9 10 6 cells mL -1 only.…”

Section: Discussionmentioning

confidence: 99%

Serum-free transfection of CHO cells with chemically defined transfection systems and investigation of their potential for transient and stable transfection

et al. 2009

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The generation of transgenic cell lines is acquired by facilitating the uptake and integration of DNA. Unfortunately, most of the systems generating stable expression systems are cost and time-consuming and transient expression is optimized to generate milligram amounts of the recombinant protein.Therefore we improved and compared two transfection systems, one based on cationic liposomes consisting of DOTAP/DOPE and the second one on polyethylenimine (PEI). Both systems have been used as chemically defined transfection systems in combination with serum-free cultivated host cell line. At first we had determined the toxicity and ideal ratio of DNA to PEI followed by determination of the optimal transfection conditions in order to achieve maximum transfection efficiency. We then directly compared DOTAP/DOPE and PEI in transient transfection experiments using enhanced green fluorescence protein (EGFP) and a human monoclonal antibody, mAb 2F5, as a model protein. The results which were achieved in case of EGFP were more than 15% transfectants at a viability of 85%. Despite the fact that expression of the mAb was found negligible we used both techniques to generate stable mAb 2F5 expressing cell lines that underwent several cycles of screening and amplification with methotrexate, and resulted in cell lines with similar volumetric production titers. These experiments serve to demonstrate the potential of stable cell lines even in case where the transient systems did not show satisfying results.

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Coexpression of acidic fibroblast growth factor enhances specific productivity and antibody titers in transiently transfected HEK293 cells

Cited by 21 publications

References 14 publications

Efficient mAb production in CHO cells incorporating PEI‐mediated transfection, mild hypothermia and the co‐expression of XBP‐1

Efficient mAb production in CHO cells incorporating PEI‐mediated transfection, mild hypothermia and the co‐expression of XBP‐1

A high‐yielding CHO transient system: Coexpression of genes encoding EBNA‐1 and GS enhances transient protein expression

Serum-free transfection of CHO cells with chemically defined transfection systems and investigation of their potential for transient and stable transfection

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