Hybrid cell line development system utilizing site-specific integration and methotrexate-mediated gene amplification in Chinese hamster ovary cells

Min, Honggi; Kim, Seul Mi; Kim, Dongwoo; Lee, Solhwi; Lee, Sumin; Lee, Jae Seong

doi:10.3389/fbioe.2022.977193

Cited by 6 publications

(4 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In the future, higher product quality, better comparability, and faster CMC timelines can be achieved by optimizing site-specific integration techniques, 21 , 22 eliminating verification runs, and simplifying comparability studies. Currently, most “fast-to-FIH” strategies are performed on standard (e.g., full-length, monospecific) mAbs owing to better understanding and control on purity.…”

Section: Discussionmentioning

confidence: 99%

Accelerating the speed of innovative anti-tumor drugs to first-in-human trials incorporating key de-risk strategies

Wang,

Quan,

Gleason

et al. 2023

mAbs

View full text Add to dashboard Cite

Pharmaceutical companies have recently focused on accelerating the timeline for initiating first-in-human (FIH) trials to allow quick assessment of biologic drugs. For example, a stable cell pool can be used to produce materials for the toxicology (Tox) study, reducing time to the clinic by 4–5 months. During the coronavirus disease 2019 (COVID-19) pandemic, the anti-COVID drugs timeline from DNA transfection to the clinical stage was decreased to 6 months using a stable pool to generate a clinical drug substrate (DS) with limited stability, virus clearance, and Tox study package. However, a lean chemistry, manufacturing, and controls (CMC) package raises safety and comparability risks and may leave extra work in the late-stage development and commercialization phase. In addition, whether these accelerated COVID-19 drug development strategies can be applied to non-COVID projects and established as a standard practice in biologics development is uncertain. Here, we present a case study of a novel anti-tumor drug in which application of “fast-to-FIH” approaches in combination with BeiGene’s de-risk strategy achieved successful delivery of a complete CMC package within 10 months. A comprehensive comparability study demonstrated that the DS generated from a stable pool and a single-cell-derived master cell bank were highly comparable with regards to process performance, product quality, and potency. This accomplishment can be a blueprint for non-COVID drug programs that approach the pace of drug development during the pandemic, with no adverse impact on the safety, quality, and late-stage development of biologics.

show abstract

Section: Discussionmentioning

confidence: 99%

Accelerating the speed of innovative anti-tumor drugs to first-in-human trials incorporating key de-risk strategies

Wang,

Quan,

Gleason

et al. 2023

mAbs

View full text Add to dashboard Cite

show abstract

“…cDNA synthesis was performed as described above, and qRT‐PCR was performed as previously described. [ 22 ] Relative gene expression levels were calculated using the ΔΔCT method and were normalized to ACTB and HPRT1 for HEK293E cells and VCL for CHO‐K1 cells. The primer sequences used are listed in Table S3.…”

Section: Methodsmentioning

confidence: 99%

“…The frozen supernatants were thawed on ice and ELISA was performed as previously described. [ 22 ] Specific mAb productivity ( q mAb ; pg/cell/day) was calculated using the mAb concentration and the EGFP‐positive integral viable cell concentration (EGFP‐IVCC).…”

Section: Methodsmentioning

confidence: 99%

Knockout of the lysosomal membrane protein, LAMP2C, improves transient gene expression in HEK293 cells via increased intracellular plasmid availability

Kim,

Lee

et al. 2023

Biotechnology Journal

Self Cite

View full text Add to dashboard Cite

Plasmid‐based transfection can be used in many applications such as transient gene expression (TGE)‐based therapeutic protein production. These applications preferentially require maximization of intracellular plasmid availability. Here, we applied a lysosome engineering approach to alleviate lysosome‐mediated nucleic acid degradation and enhance the TGE in mammalian cells. By knocking out the lysosomal membrane protein LAMP2C, which is known to be the main player in RNautophagy/DNautophagy (RDA), we significantly improved transient fluorescent protein expression in HEK293 cells by improving the retention rate of transfected plasmids; however, this effect was not observed in CHO cells. Additional knockout of a lysosomal membrane transporter and another RDA player, SIDT2, was ineffective, regardless of the presence of LAMP2C. LAMP2C knockout enhanced TGE‐based mAb production in HEK293 cells by up to 2.82‐fold increase in specific mAb productivity. Taken together, these results demonstrate that HEK293 cells can be engineered to improve the usage of the transfected plasmid via knockout of the lysosomal membrane protein LAMP2C and provide efficient host cells in TGE systems for therapeutic protein production.

show abstract

“…With gene exchange by the RMCE method, DNA breaks and rejoins occur only between the corresponding loci, resulting in protein-producing cell lines stably expressed at fixed loci ( Kameyama et al, 2010 ). Many site-specific recombinase systems have been attempted to mediate expression cassette exchange by site-specific recombination in mammalian cells, e.g., Cre/ lox , Flp/ FRT , BxB1 recombinase, and PhiC31 integrase ( Zhang et al, 2015 ; Inniss et al, 2017 ; Pourtabatabaei et al, 2021 ; Min et al, 2022 ). Among the studied and employed recombinases, Cre recombinase stands out as one of the most intensively investigated ones, exhibiting higher site specificity and efficiency ( Turan et al, 2013 ).…”

Section: Introductionmentioning

confidence: 99%

Construction and application of a multifunctional CHO cell platform utilizing Cre/lox and Dre/rox site-specific recombination systems

Zhang,

Chang,

Miao

et al. 2023

Front. Bioeng. Biotechnol.

View full text Add to dashboard Cite

During the development of traditional Chinese hamster ovary (CHO) cell lines, target genes randomly integrate into the genome upon entering the nucleus, resulting in unpredictable productivity of cell clones. The characterization and screening of high-yielding cell lines is a time-consuming and expensive process. Site-specific integration is recognized as an effective approach for overcoming random integration and improving production stability. We have designed a multifunctional expression cassette, called CDbox, which can be manipulated by the site-specific recombination systems Cre/lox and Dre/rox. The CDbox expression cassette was inserted at the Hipp11(H11) locus hotspot in the CHO-K1 genome using CRISPR/Cas9 technology, and a compliant CHO-CDbox cell platform was screened and obtained. The CHO-CDbox cell platform was transformed into a pool of EGFP-expressing cells using Cre/lox recombinase-mediated cassette exchange (RMCE) in only 2 weeks, and this expression remained stable for at least 75 generations without the need for drug stress. Subsequently, we used the Dre/rox system to directly eliminate the EGFP gene. In addition, two practical applications of the CHO-CDbox cell platform were presented. The first was the quick construction of the Pembrolizumab antibody stable expression strain, while the second was a protocol for the integration of surface-displayed and secreted antibodies on CHO cells. The previous research on site-specific integration of CHO cells has always focused on the single functionality of insertion of target genes. This newly developed CHO cell platform is expected to offer expanded applicability for protein production and gene function studies.

show abstract

Hybrid cell line development system utilizing site-specific integration and methotrexate-mediated gene amplification in Chinese hamster ovary cells

Cited by 6 publications

References 40 publications

Accelerating the speed of innovative anti-tumor drugs to first-in-human trials incorporating key de-risk strategies

Accelerating the speed of innovative anti-tumor drugs to first-in-human trials incorporating key de-risk strategies

Knockout of the lysosomal membrane protein, LAMP2C, improves transient gene expression in HEK293 cells via increased intracellular plasmid availability

Construction and application of a multifunctional CHO cell platform utilizing Cre/lox and Dre/rox site-specific recombination systems

Contact Info

Product

Resources

About