The use of site-specific recombination and cassette exchange technologies for monoclonal antibody production in Chinese Hamster ovary cells: retrospective analysis and future directions

Srirangan, Kajan; Loignon, Martin; Durocher, Yves

doi:10.1080/07388551.2020.1768043

Cited by 23 publications

(21 citation statements)

References 89 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…9 Site-specific integration (SSI) has been used by several groups to reduce the variability in recombinant cell lines, enabling process simplification and predictability. 10 SSI is facilitated by engineered landing pads which are exogenous sequences introduced into the host cell chromosomal DNA that contain recombination sites for the integration of expression cassettes by recombinase-mediated cassette exchange (RMCE). 11 Landing pads located in the genomes of SSI competent host cell lines utilize recombination target sites derived from site specific recombinase systems such as the Saccharomyces cerevisiae-derived Flp-Frt system or the bacteriophage P1 derived Cre-loxP system.…”

mentioning

confidence: 99%

CHOK1SV GS‐KO SSIexpression system: A combination of theFer1L4locus and glutamine synthetase selection

Feary

Moffat

Casperson

et al. 2021

Biotechnol Progress

View full text Add to dashboard Cite

There are an ever-increasing number of biopharmaceutical candidates in clinical trials fueling an urgent need to streamline the cell line development process. A critical part of the process is the methodology used to generate and screen candidate cell lines compatible with GMP manufacturing processes. The relatively large amount of clone phenotypic variation observed from conventional "random integration" (RI)-based cell line construction is thought to be the result of a combination of the position variegation effect, genome plasticity and clonal variation. Site-specific integration (SSI) has been used by several groups to temper the influence of the position variegation effect and thus reduce variability in expression of biopharmaceutical candidates.Following on from our previous reports on the application of the Fer1L4 locus for SSI in CHOK1SV (10E9), we have combined this locus and a CHOK1SV glutamine synthetase knockout (GS-KO) host to create an improved expression system. The host, CHOK1SV GS-KO SSI (HD7876), was created by homology directed integration of a targetable landing pad flanked with incompatible Frt sequences in the Fer1L4 gene.The targeting vector contains a promoterless GS expression cassette and monoclonal antibody (mAb) expression cassettes, flanked by Frt sites compatible with equivalent sites flanking the landing pad in the host cell line. SSI clones expressing four antibody candidates, selected in a streamlined cell line development process, have mAb titers which rival RI (1.0-4.5 g/L) and robust expression stability (100% of clones stable through the 50 generation "manufacturing window" which supports commercial manufacturing at 12,000 L bioreactor scale).

show abstract

mentioning

confidence: 99%

CHOK1SV GS‐KO SSIexpression system: A combination of theFer1L4locus and glutamine synthetase selection

Feary

Moffat

Casperson

et al. 2021

Biotechnol Progress

View full text Add to dashboard Cite

show abstract

“…Such proteins offer enormous advances in disease therapy (or diagnosis) and are products of high commercial value. Over the last several decades there have been significant advances in the efficiency of CHO cells to produce these recombinant proteins (Budge et al, 2020;Dreesen & Fussenegger, 2011;Kunert & Reinhart, 2016;Sharker & Rahman, 2020;Srirangan et al, 2020). However, the rate of protein production remains a significant bottleneck, particularly for many of the novel format based biotherapeutics now in development, and therefore there remains substantial interest in further advancing the productivity of CHO cells (Budge et al, 2020;Sharker & Rahman, 2020).…”

Section: Introductionmentioning

confidence: 99%

Constitutively active Rheb mutants [T23M] and [E40K] drive increased production and secretion of recombinant protein in Chinese hamster ovary cells

Poi

Xie

Smales

et al. 2021

Biotech & Bioengineering

View full text Add to dashboard Cite

Monoclonal antibodies (mAbs) are high value agents used for disease therapy ("biologic drugs") or as diagnostic tools which are widely used in the healthcare sector. They are generally manufactured in mammalian cells, in particular Chinese hamster ovary (CHO) cells cultured in defined media, and are harvested from the medium. Rheb is a small GTPase which, when bound to GTP, activates mechanistic target of rapamycin complex 1, a protein kinase that drives anabolic processes including protein synthesis and ribosome biogenesis. Here, we show that certain constitutively active mutants of Rheb drive faster protein synthesis in CHO cells and increase the expression of proteins involved in the processing of secreted proteins in the endoplasmic reticulum, which expands in response to expression of Rheb mutants. Active Rheb mutants, in particular Rheb[T23M], drive increased cell number under serum-free conditions similar to those used in the biotechnology industry. Rheb[T23M] also enhances the expression of the reporter protein luciferase and, especially strongly, the secreted Gaussia luciferase. Moreover, Rheb [T23M] markedly (2-3 fold) enhances the amount of this luciferase and of a model immunoglobulin secreted into the medium. Our data clearly demonstrate that expressing Rheb[T23M] in CHO cells provides a simple approach to promoting their growth in defined medium and the production of secreted proteins of high commercial value.

show abstract

“…Site‐specific integration (SSI) technology has brought forward much greater efficiency in inserting genes of interest (GOIs) into the host cell genome than the classical random integration transfection methods. While SSI has improved the productivity of the stable cell lines, the much higher efficiency of recombinant gene integration into the genome of the host cells than the traditional random insertion is more attractive to the industry 26–33 . While the traditional method of plating and screening of stable cell pools usually takes 2 months, the SSI stable pool takes as short as 1–2 weeks by skipping the labor‐intensive mini‐pool screening, due to enhanced probability of genomic integration 34–37 .…”

Section: Methodsmentioning

confidence: 99%

Reshaping cell line development and CMC strategy for fast responses to pandemic outbreak

Zhang

Wang

et al. 2021

Biotechnology Progress

View full text Add to dashboard Cite

The global pandemic outbreak COVID‐19 (SARS‐COV‐2), has prompted many pharmaceutical companies to develop vaccines and therapeutic biologics for its prevention and treatment. Most of the therapeutic biologics are common human IgG antibodies, which were identified by next‐generation sequencing (NGS) with the B cells from the convalescent patients. To fight against pandemic outbreaks like COVID‐19, biologics development strategies need to be optimized to speed up the timeline. Since the advent of therapeutic biologics, strategies of transfection and cell line selection have been continuously improved for greater productivity and efficiency. NGS has also been implemented for accelerated cell bank testing. These recent advances enable us to rethink and reshape the chemistry, manufacturing, and controls (CMC) strategy in order to start supplying Good Manufacturing Practices (GMP) materials for clinical trials as soon as possible. We elucidated an accelerated CMC workflow for biologics, including using GMP‐compliant pool materials for phase I clinical trials, selecting the final clone with product quality similar to that of phase I materials for late‐stage development and commercial production.

show abstract

The use of site-specific recombination and cassette exchange technologies for monoclonal antibody production in Chinese Hamster ovary cells: retrospective analysis and future directions

Cited by 23 publications

References 89 publications

CHOK1SV GS‐KO SSIexpression system: A combination of theFer1L4locus and glutamine synthetase selection

CHOK1SV GS‐KO SSIexpression system: A combination of theFer1L4locus and glutamine synthetase selection

Constitutively active Rheb mutants [T23M] and [E40K] drive increased production and secretion of recombinant protein in Chinese hamster ovary cells

Reshaping cell line development and CMC strategy for fast responses to pandemic outbreak

Contact Info

Product

Resources

About