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

Backliwal, Gaurav; Hildinger, Markus; Chenuet, Sébastien; Wulhfard, Sarah; Jesús, María de; Wurm, Florian M.

doi:10.1093/nar/gkn423

Cited by 204 publications

(166 citation statements)

References 21 publications

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“…Our SINEUP system adds to those already established for optimization of mAb production. 11-13 The combination of SINEUP technology with existing and upcoming technologies will be evaluated in the future, to verify whether optimal mAb production can be reached, in terms of even more pronounced yields and unaltered PTMs.…”

Section: Resultsmentioning

confidence: 99%

A long non-coding SINEUP RNA boosts semi-stable production of fully human monoclonal antibodies in HEK293E cells

Sasso

Latino

Froechlich

et al. 2018

mAbs

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Use of monoclonal antibodies is emerging as a highly promising and fast-developing scenario for innovative treatment of viral, autoimmune and tumour diseases. The search for diagnostic and therapeutic antibodies currently depends on in vitro screening approaches, such as phage and yeast display technologies. Antibody production still represents a critical step for preclinical and clinical evaluations. Accordingly, improving production of monoclonal antibodies represents an opportunity, to facilitate downstream target validations. SINEUP RNAs are long non-coding transcripts, possessing the ability to enhance translation of selected mRNAs. We applied SINEUP technology to semi-stable production of monoclonal antibodies in HEK293E cells, which allows for episomal propagation of the expression vectors encoding the heavy and light chains of IgGs. Co-expression of SINEUP RNA with mRNAs encoding heavy and light chains of IgG4s was able to increase the production of different anti-CLDN1 antibodies up to three-fold. Improved production of monoclonal antibodies was achieved both in transiently transfected HEK293E cells and in cellular clones with stable expression of the SINEUP. Compared to antibody preparations obtained under standard conditions, the anti-CLDN1 IgG4s produced in the presence of the SINEUP transcript showed unaltered post-translational modifications, and retained the ability to recognize their target. We thus propose SINEUP technology as a valuable tool to enhance semi-stable antibody production in human cell lines.

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Section: Resultsmentioning

confidence: 99%

A long non-coding SINEUP RNA boosts semi-stable production of fully human monoclonal antibodies in HEK293E cells

Sasso

Latino

Froechlich

et al. 2018

mAbs

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“…The same three expression vectors were then used to determine the effect of temperatures other than 31°C on TGE. Cells were transfected as described above and shifted to 29,31, or 33°C at 4 h post-transfection. At all three temperatures tested, there was enhancement of antibody and GFP production relative to the control culture at 37°C, with the highest yields of GFP ( Figure 2A) and antibody ( Figure 2B) at 31 and 33°C, respectively.…”

Section: Transient Gene Expression Under Mild Hypothermicmentioning

confidence: 99%

Mild Hypothermia Improves Transient Gene Expression Yields Several Fold in Chinese Hamster Ovary Cells

Wulhfard

Tissot

Bouchet

et al. 2008

Biotechnology Progress

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Large-scale transient gene expression (TGE) in mammalian cells is a rapid method to generate recombinant proteins, but the volumetric productivity for secreted proteins is still more than an order of magnitude lower than the yields typically achieved with recombinant cell lines. Here transient recombinant protein production in Chinese hamster ovary cells transfected with linear 25 kDa polyethylenimine was significantly enhanced by incubation of the cells at temperatures ranging from 29 to 33°C after DNA delivery. With this approach, transient recombinant antibody yields of 60-80 mg/L were achieved within 6 days of transfection. The increase in TGE correlated with the accumulation of cells in the G1 phase of the cell cycle, increased cell size, higher cell viability, higher steady-state levels of transgene mRNA, reduced consumption of nutrients, and decreased accumulation of waste products. The enhancement of TGE was not vector-dependent, but the presence of the woodchuck hepatitis virus post-transcriptional regulatory element in the 3′ untranslated region of the transgene mRNA increased transient recombinant antibody expression more than 3-fold at 31°C as compared to expression at 37°C. The yields achieved by the low-temperature enhancement of TGE in CHO cells makes this technology feasible for the rapid production of gram amounts of secreted recombinant proteins at large scale (up to 100 L).

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“…However, studies testing serum versus serum-free media have shown that higher titers could be obtained in serum-containing media (Durocher et al 2002), which suggests that there may be components in serum-free media that inhibit PEI-mediated transient transfection or that serum-free media lacks components necessary for successful transfections. Despite this observation, there have been reports of serum-free medium used with both 293 and CHO cells that allow levels of recombinant protein expression in the range of hundreds of milligrams per liter, with a recent study reporting over 1 g/L of recombinant monoclonal antibody produced in 293 cells (Backliwal et al 2008). In this study, we have examined several different media for their compatibility with PEI-mediated transient transfection in CHO cells.…”

Section: Introductionmentioning

confidence: 99%

Iron (III) citrate inhibits polyethylenimine-mediated transient transfection of Chinese hamster ovary cells in serum-free medium

Eberhardy¹,

Radzniak²,

Zhong³

2009

Cytotechnology

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Recent advances in transient transfection protocols using polyethylenimine (PEI) as a transfection reagent have led to the development of economical methods that provide yields sufficient for industrial production of proteins for many preclinical needs. There are many variables that can be optimized to improve protein expression in transient transfection, and one of the most critical is the medium in which the cells are grown. While transfection with PEI works well in media containing serum, the biopharmaceutical industry is moving away from animal-derived components in media. A number of serum-free media have been found to allow transient transfection, but many others do not for reasons that are not clear. Thus, knowledge of the components of serum-free media that can cause inhibition of PEI-mediated transient transfection would be useful for media development. In this study, an analysis was performed of various components of a serum-free medium used for Chinese hamster ovary cells in which PEI-mediated transient transfection was inhibited. We found that an iron supplement added to the medium was responsible for the inhibition. Further investigation showed that iron (III) citrate, a common iron chelator found in serumfree medium, was the specific component that caused the effect. Further, we showed that inhibition of transient transfection was caused by iron (III) citrate specifically, rather than citrate or iron alone. Finally, we showed that various iron chelators in serum-free media other than iron (III) citrate do not inhibit antibody expression.

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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

Cited by 204 publications

References 21 publications

A long non-coding SINEUP RNA boosts semi-stable production of fully human monoclonal antibodies in HEK293E cells

A long non-coding SINEUP RNA boosts semi-stable production of fully human monoclonal antibodies in HEK293E cells

Mild Hypothermia Improves Transient Gene Expression Yields Several Fold in Chinese Hamster Ovary Cells

Iron (III) citrate inhibits polyethylenimine-mediated transient transfection of Chinese hamster ovary cells in serum-free medium

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