Accelerating stable recombinant cell line development by targeted integration

Rehberger, Bernd; Wodarczyk, Claas; Reichenbächer, Britta; Köhler, Janet; Weber, R. C.; Müller, Dethardt

doi:10.1186/1753-6561-7-s6-p111

Cited by 15 publications

(15 citation statements)

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“…For all practical purposes even if a reverse rate existed it does not hinder efficiently creating SSI pools where clearly the equilibrium favors integration over excision, as we observe ~91% exchange by flow cytometry. The data are comparable to 90–99% GFP negative, gene of interest expressing cells claimed to be observed with the TurboCell CHO‐K1 SSI expression system 24 …”

Section: Resultssupporting

confidence: 77%

“…The data are comparable to 90-99% GFP negative, gene of interest expressing cells claimed to be observed with the TurboCell CHO-K1 SSI expression system. 24…”

Section: Rmce Using the Chok1sv Gs-ko Ssi Hostmentioning

confidence: 99%

“…[13][14][15][16][17][18][19][20][21] Hamaker and Lee, 22 have hypothesized that SSI hosts would give a throughput advantage over RI hosts by reducing the number of clones that need to be screened to identify a cell line with suitable growth, stability, and expression characteristics. These claims are starting to be supported in recent examples of SSI systems being developed in the sector (ThermoFisher Scientific [Flp-In-CHO 23 ] Rentschler Biotechnologie [TurboCell 24 ], Novo Nordisk Foundation Center [isoCHO 25 ], and Genentech [TI host 26 ]).…”

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confidence: 99%

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

Feary

Moffat

Casperson

et al. 2021

Biotechnol Progress

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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).

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Section: Resultssupporting

confidence: 77%

“…The data are comparable to 90-99% GFP negative, gene of interest expressing cells claimed to be observed with the TurboCell CHO-K1 SSI expression system. 24…”

Section: Rmce Using the Chok1sv Gs-ko Ssi Hostmentioning

confidence: 99%

mentioning

confidence: 99%

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

Feary

Moffat

Casperson

et al. 2021

Biotechnol Progress

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“…A Chinese hamster ovary (CHO) cell line producing an IgG monoclonal antibody was used for the experiments. The cell line is Rentschler Biopharma’s proprietary mammalian cell line which is a pre-tagged CHO K1 master cell line for directed integration of the gene of interest by recombinase mediated cassette exchange (RMCE) ( Rehberger et al, 2013 ).…”

Section: Methodsmentioning

confidence: 99%

Time-Resolved Monitoring of the Oxygen Transfer Rate of Chinese Hamster Ovary Cells Provides Insights Into Culture Behavior in Shake Flasks

Ihling

Munkler

Berg

et al. 2021

Front. Bioeng. Biotechnol.

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Cultivations of mammalian cells are routinely conducted in shake flasks. In contrast to instrumented bioreactors, reliable options for non-invasive, time-resolved monitoring of the culture status in shake flasks are lacking. The Respiration Activity Monitoring Respiration Activity Monitoring System system was used to determine the oxygen transfer rate (OTR) in shake flasks. It was proven that the OTR could be regarded as equal to the oxygen uptake rate as the change of the dissolved oxygen concentration in the liquid phase over time was negligibly small. Thus, monitoring the oxygen transfer rate (OTR) was used to increase the information content from shake flask experiments. The OTR of a Chinese hamster ovary cell line was monitored by applying electrochemical sensors. Glass flasks stoppered with cotton plugs and polycarbonate flasks stoppered with vent-caps were compared in terms of mass transfer characteristics and culture behavior. Similar mass transfer resistances were determined for both sterile closures. The OTR was found to be well reproducible within one experiment (standard deviation <10%). It correlated with changes in cell viability and depletion of carbon sources, thus, giving more profound insights into the cultivation process. Culture behavior in glass and polycarbonate flasks was identical. Monitoring of the OTR was applied to a second culture medium. Media differed in the maximum OTR reached during cultivation and in the time when all carbon sources were depleted. By applying non-invasive, parallelized, time-resolved monitoring of the OTR, the information content and amount of data from shake flask experiments was significantly increased compared to manual sampling and offline analysis. The potential of the technology for early-stage process development was demonstrated.

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“…Data for the individual AAs are shown in Tables S1 and S2. Methods A and B were applied to analyze AAs during the cultivation of CHO cells (TurboCell TM [10]; Rentschler Biopharma, Laupheim, Germany) in a 4-L reactor (Belach Bioteknik, Stockholm, Sweden). The samples were kindly provided by the division of Bioprocess Design, Department of Industrial Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden.…”

Section: Tůma Et Al Previously Developed Ce-c 4 D Methodsmentioning

confidence: 99%

Analysis of amino acids in cell culture supernatant using capillary electrophoresis with contactless conductivity detection

Mikkonen

Emmer

2021

Electrophoresis

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CE-C 4 D methods for the analysis of amino acids (AAs) are presented. Combining the results from two methods with acetic acid and cyclodextrin-based BGEs, 20 proteinogenic AAs could be analyzed using CE. CE-C 4 D was also, for the first time, applied to analyze free AAs in samples of mammalian cell culture supernatant. After dilution as only sample preparation, combining the results of the two CE methods allowed monitoring the concentration changes of 17 AAs in samples taken during the cultivation of CHO cells.

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Accelerating stable recombinant cell line development by targeted integration

Cited by 15 publications

References 0 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

Time-Resolved Monitoring of the Oxygen Transfer Rate of Chinese Hamster Ovary Cells Provides Insights Into Culture Behavior in Shake Flasks

Analysis of amino acids in cell culture supernatant using capillary electrophoresis with contactless conductivity detection

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