Development of Transfection and High-Producer Screening Protocols for the CHOK1SV Cell System

Edmonds, M. Celina de la Cruz; Tellers, Melanie; Chan, Christine P.; Salmon, Peter; Robinson, David K.; Markusen, Julia F.

doi:10.1385/mb:34:2:179

Cited by 43 publications

(9 citation statements)

References 24 publications

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“…Due to the short timeline of the cell line generation, a GS-based MSX-selection system has been increasingly used for the establishment of rCHO cell lines. The MSX concentrations used for a single round of selection are usually 10–50 μM MSX for wild type CHO cell lines such as CHO-K1 and CHOK1SV 9 , 11 , 19 , 24 , 25 and 0–25 μM for GS-knockout CHO cell lines with an improved selection stringency 12 , 13 . However, despite its popularity, the characterization of the GS-based selection system has not been fully substantiated yet.…”

Section: Discussionmentioning

confidence: 99%

Comprehensive characterization of glutamine synthetase-mediated selection for the establishment of recombinant CHO cells producing monoclonal antibodies

Noh

Shin²,

Lee

2018

Sci Rep

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To characterize a glutamine synthetase (GS)-based selection system, monoclonal antibody (mAb) producing recombinant CHO cell clones were generated by a single round of selection at various methionine sulfoximine (MSX) concentrations (0, 25, and 50 μM) using two different host cell lines (CHO-K1 and GS-knockout CHO). Regardless of the host cell lines used, the clones selected at 50 μM MSX had the lowest average specific growth rate and the highest average specific production rates of toxic metabolic wastes, lactate and ammonia. Unlike CHO-K1, high producing clones could be generated in the absence of MSX using GS-knockout CHO with an improved selection stringency. Regardless of the host cell lines used, the clones selected at various MSX concentrations showed no significant difference in the GS, heavy chain, and light chain gene copies (P > 0.05). Furthermore, there was no correlation between the specific mAb productivity and these three gene copies (R2 ≤ 0.012). Taken together, GS-mediated gene amplification does not occur in a single round of selection at a MSX concentration up to 50 μM. The use of the GS-knockout CHO host cell line facilitates the rapid generation of high producing clones with reduced production of lactate and ammonia in the absence of MSX.

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Section: Discussionmentioning

confidence: 99%

Comprehensive characterization of glutamine synthetase-mediated selection for the establishment of recombinant CHO cells producing monoclonal antibodies

Noh

Shin²,

Lee

2018

Sci Rep

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“…The transfection vector utilizes the glutamine synthetase (GS) gene expression system. The transfected CHOK1SV expresses the GS enzyme endogenously; thus, positive transfectants can only be selected in the presence of Methionine Sulfoximine (MSX), an inhibitor of the GS enzyme and glutamine-free media (Edmonds et al, 2006). The suspension-adapted CHOK1SV cell line (licensed from Lonza Biologics) was used as the host to express an IgG4 recombinant protein using the GS expression vector system (licensed from Lonza Biologics) in this study.…”

Section: Methodsmentioning

confidence: 99%

Novel micro‐bioreactor high throughput technology for cell culture process development: Reproducibility and scalability assessment of fed‐batch CHO cultures

Amanullah¹,

Otero²,

Mikola³

et al. 2010

Biotech & Bioengineering

102

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With increasing timeline pressures to get therapeutic and vaccine candidates into the clinic, resource intensive approaches such as the use of shake flasks and bench-top bioreactors may limit the design space for experimentation to yield highly productive processes. The need to conduct large numbers of experiments has resulted in the use of miniaturized high-throughput (HT) technology for process development. One such high-throughput system is the SimCell platform, a robotically driven, cell culture bioreactor system developed by BioProcessors Corp. This study describes the use of the SimCell micro-bioreactor technology for fed-batch cultivation of a GS-CHO transfectant expressing a model IgG4 monoclonal antibody. Cultivations were conducted in gas-permeable chambers based on a micro-fluidic design, with six micro-bioreactors (MBs) per micro-bioreactor array (MBA). Online, non-invasive measurement of total cell density, pH and dissolved oxygen (DO) was performed. One hundred fourteen parallel MBs (19 MBAs) were employed to examine process reproducibility and scalability at shake flask, 3- and 100-L bioreactor scales. The results of the study demonstrate that the SimCell platform operated under fed-batch conditions could support viable cell concentrations up to least 12 x 10(6) cells/mL. In addition, both intra-MB (MB to MB) as well as intra-MBA (MBA to MBA) culture performance was found to be highly reproducible. The intra-MB and -MBA variability was calculated for each measurement as the coefficient of variation defined as CV (%) = (standard deviation/mean) x 100. The % CV values for most intra-MB and intra-MBA measurements were generally under 10% and the intra-MBA values were slightly lower than those for intra-MB. Cell growth, process parameters, metabolic and protein titer profiles were also compared to those from shake flask, bench-top, and pilot scale bioreactor cultivations and found to be within +/-20% of the historical averages.

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“…The effects of selection pressure on colony growth and number of colonies obtained during the initial selection phase in cell line development has previously been demonstrated for MSX concentrations of 25 and 50 lM (de la Cruz Edmonds et al 2006). In our study, the effect of an even higher concentration of MSX selection agent following transfection was examined in terms of number of colonies obtained and titer distribution in primary and secondary screens.…”

Section: Resultsmentioning

confidence: 94%

Automation of cell line development

et al. 2009

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An automated platform for development of high producing cell lines for biopharmaceutical production has been established in order to increase throughput and reduce development costs. The concept is based on the Cello robotic system (The Automation Partnership) and covers screening for colonies and expansion of static cultures. In this study, the glutamine synthetase expression system (Lonza Biologics) for production of therapeutic monoclonal antibodies in Chinese hamster ovary cells was used for evaluation of the automation approach. It is shown that the automated procedure is capable of producing cell lines of equal quality to the traditionally generated cell lines in terms of colony detection following transfection and distribution of IgG titer in the screening steps. In a generic fed-batch evaluation in stirred tank bioreactors, IgG titers of 4.7 and 5.0 g/L were obtained for best expressing cell lines. We have estimated that the number of completed cell line development projects can be increased up to three times using the automated process without increasing manual workload, compared to the manual process. Correlation between IgG titers obtained in early screens and titers achieved in fed-batch cultures in shake flasks was found to be poor. This further implies the benefits of utilizing a high throughput system capable of screening and expanding a high number of transfectants. Two concentrations, 56 and 75 lM, of selection agent, methionine sulphoximine (MSX), were applied to evaluate the impact on the number of colonies obtained post transfection. When applying selection medium containing 75 lM MSX, fewer low producing transfectants were obtained, compared to cell lines selected with 56 lM MSX, but an equal number of high producing cell lines were found. By using the higher MSX concentration, the number of cell line development projects run in parallel could be increased and thereby increasing the overall capacity of the automated platform process.

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Development of Transfection and High-Producer Screening Protocols for the CHOK1SV Cell System

Cited by 43 publications

References 24 publications

Comprehensive characterization of glutamine synthetase-mediated selection for the establishment of recombinant CHO cells producing monoclonal antibodies

Comprehensive characterization of glutamine synthetase-mediated selection for the establishment of recombinant CHO cells producing monoclonal antibodies

Novel micro‐bioreactor high throughput technology for cell culture process development: Reproducibility and scalability assessment of fed‐batch CHO cultures

Automation of cell line development

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