Transcriptional and post-transcriptional targeting for enhanced transient gene expression in CHO cells

Rajendra, Yashas; Hougland, Maria D.; Schmitt, Matthew G.; Barnard, Gavin C.

doi:10.1007/s10529-015-1938-6

Cited by 18 publications

(17 citation statements)

References 20 publications

(34 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…On the day of transfection, cells were recovered by centrifugation at 450 g for 3 min and re‐suspended in LM‐TNX medium (identical to LM‐Growth medium minus ferric ammonium citrate) at 4 × 10 6 cells/mL. DNA (3.2 mg/L) and PEI (8 mg/L) were sequentially added to the cells as previously described . Cultures were fed at 4 h post‐transfection.…”

Section: Methodsmentioning

confidence: 99%

Generation of stable Chinese hamster ovary pools yielding antibody titers of up to 7.6 g/L using the piggyBac transposon system

Rajendra

Peery

Barnard

2016

Biotechnology Progress

Self Cite

View full text Add to dashboard Cite

Chinese hamster ovary (CHO) cells remain the default production host for many biopharmaceutical drugs, particularly monoclonal antibodies (mAb). Production of gram and kilogram quantities of protein typically requires the generation of stable CHO clones. Unfortunately, this process takes several months, significantly slowing down the drug discovery and development process. Therefore, improved technologies are needed to accelerate biopharmaceutical drug discovery and final drug substance manufacturing. In this study, we describe the generation of stable CHO pools using the piggyBac transposon system. We evaluated the system using four model antibody molecules (3 mAbs and 1 bispecific Ab). Stable CHO pools were isolated in 7-12 days. Using a simple 16-day fed-batch process, we measured titers ranging from 2.3 to 7.6 g/L for the four model antibodies. This represented a 4- to 12-fold increase relative to the controls. Additionally, we isolated stable CHO clones. We found that the stable CHO clones isolated from the piggyBac transposon pools yielded titers two to threefold higher relative to the control clones. Taken together, these results suggest that stable CHO pool and clone generation can be significantly improved by using the piggyBac transposon system. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1301-1307, 2016.

show abstract

Section: Methodsmentioning

confidence: 99%

Generation of stable Chinese hamster ovary pools yielding antibody titers of up to 7.6 g/L using the piggyBac transposon system

Rajendra

Peery

Barnard

2016

Biotechnology Progress

Self Cite

View full text Add to dashboard Cite

show abstract

“…We wanted to evaluate the expression and assembly of hetero-mAbs in our transient CHO platform which utilizes the same cell line and media package used for stable CHO manufacturing. 12 As a starting point, the dual vectors coding for each half of the four different hetero-mAbs were used for the expression analysis in transient CHO. Table 1 lists the four hetero-mAbs used in the study and corresponding dual vector names for ease of understanding.…”

Section: Resultsmentioning

confidence: 99%

“…Transfections were performed as described previously . In brief, 2 days prior to transfection, CHO cells were seeded at 1.5 × 10 6 cells/ml in LM‐growth media.…”

Section: Methodsmentioning

confidence: 99%

“…Transfections were performed as described previously. 12 In brief, 2 days prior to transfection, CHO cells were seeded at 1.5 3 10 6 cells/ml in LM-growth media. On the day of transfection, the cells were recovered by centrifugation at 450g for 3 min and re-suspended in LM-TNX media (identical to LM-Growth medium minus ferric ammonium citrate plus 0.125% N, N Dimethylacetamide) at 4 3 10 6 cells/mL.…”

Section: Transient Transfectionmentioning

confidence: 99%

“…Expression levels were assessed using Fortebio-Octet on day 14. Cultures were harvested and 4 mL of each culture was transferred to a 24DWP and subjected to semiautomatic robot based protein A purification as previously described (Barnard et al, 2015). Purified protein was analyzed on Agilent 2100 Bioanalyzer system using the Agilent Protein 230 Kit.…”

Section: Stable Cho Expression From Single Vectors (Quad Vectors)mentioning

confidence: 99%

See 2 more Smart Citations

Transient and stable CHO expression, purification and characterization of novel hetero‐dimeric bispecific IgG antibodies

Rajendra

Peery

Hougland

et al. 2017

Biotechnology Progress

Self Cite

View full text Add to dashboard Cite

IgG bispecific antibodies (BsAbs) represent one of the preferred formats for bispecific antibody therapeutics due to their native-like IgG properties and their monovalent binding to each target. Most reported studies utilized transient expression in HEK293 cells to produce BsAbs. However, the expression of biotherapeutic molecules using stable CHO cell lines is commonly used for biopharmaceutical manufacturing. Unfortunately, limited information is available in the scientific literature on the expression of BsAbs in CHO cell lines. In this study we describe an alternative approach to express the multiple components of IgG BsAbs using a single plasmid vector (quad vector). This single plasmid vector contains both heavy chain genes and both light chain genes required for the expression and assembly of the IgG BsAb, along with a selectable marker. We expressed, purified, and characterized four different IgG BsAbs or "hetero-mAbs" using transient CHO expression and stable CHO minipools. Transient CHO titers ranged from 90 to 160 mg/L. Stable CHO titers ranged from 0.4 to 2.3 g/L. Following a simple Protein A purification step, the percentage of correctly paired BsAbs ranged from 74% to 98% as determined by mass spectrometry. We also found that information generated from transient CHO expression was similar to information generated using stable CHO minipools. In conclusion, the quad vector approach represents a simple, but effective, alternative approach for the generation of IgG BsAbs in both transient CHO and stable CHO expression systems. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:469-477, 2017.

show abstract

Transient Gene Expression‐Based Protein Production in Recombinant Mammalian Cells

Lee

Hansen

Park

et al. 2019

Cell Culture Engineering

View full text Add to dashboard Cite

Transcriptional and post-transcriptional targeting for enhanced transient gene expression in CHO cells

Abstract: Transcriptional and post-transcriptional pathways of transient gene expression can be targeted to increase titers without resorting to host cell line engineering in a simple, short, 7 day production process.

Cited by 18 publications

References 20 publications

Generation of stable Chinese hamster ovary pools yielding antibody titers of up to 7.6 g/L using the piggyBac transposon system

Generation of stable Chinese hamster ovary pools yielding antibody titers of up to 7.6 g/L using the piggyBac transposon system

Transient and stable CHO expression, purification and characterization of novel hetero‐dimeric bispecific IgG antibodies

Transient Gene Expression‐Based Protein Production in Recombinant Mammalian Cells

Contact Info

Product

Resources

About