Freedom™ DG44 Kit - FAQs

我们推荐您在建立稳转细胞系之前，将每一蛋白亚基克隆分别克隆至两个载体中，以确定何种载体组合能够在瞬转条件下提供最高的蛋白产量。

尽管MTX扩增的机理尚不十分清楚，不过人们认为这两个载体会趋向整合于相同的基因组位点，因此也会实现同步扩增。

在小规模的起始实验中，G418的存在不会对蛋白生产造成影响。不过，我们并不推荐在大规模蛋白生产过程中（即在生物反应器中）添加G418。

We recommend cloning each protein subunit into each of the vectors to determine which vector combination gives the highest protein yield in transient transfection before making the stable cell line.

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Even though the exact mechanism for MTX amplification is not well understood, the thinking is that both vectors tend to integrate in the same genomic locus and hence would get amplified at the same time.

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Using G418 should not pose a problem for low-scale protein production for initial studies. However, we do not recommend adding G418 during large-scale protein production (i.e., in a bioreactor).

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There are a couple of concerns associated with high MTX concentrations. One is the stability of the cell line generated using high MTX concentrations. The other is that it may be amplifying the DFHR gene without amplifying the gene of interest along with it stoichiometrically. We recommend the more conservative approach of 4 µM MTX for amplification.

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To inquire about a Commercial Production or Service License, please email: outlicensing@thermofisher.com.

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Yes. Contact our field-based Process Science Fellows (PSFs) at pd.direct@lifetech.com for technical support.

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To inquire about a commercial license, please email: pd.direct@lifetech.com. To read the complete list of FAQs related to licensing, go here (https://www.thermofisher.com/us/en/home/life-science/bioproduction/bp-misc/gibco-freedom/freedom-cho-s-cells-and-freedom-dg44-cells-licensing.html).

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No. Only a one-time license fee is required, and it covers the commercialization of an unlimited number of molecules using the CHO DG44 Cells (cGMP banked).

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A commercial use license is required prior to the sale of expressed proteins, or when an IND submission is being prepared for first in human clinical studies.

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IgG-expressing CHO DG44 clones readily scale-up from shake flask to bioreactor vessels while maintaining growth and titer performance.

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When using limiting dilution cloning, careful evaluation of monoclonality of a clone is required before assessing stability of expression in order to avoid heterogeneous clones that can drift into separate populations and appear unstable.

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Only two transfections are recommended.

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Both FreeStyle MAX Reagent (included in the kit) and the Neon Transfection System provide stable pools with similar final titers

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Good results can be obtaining using either limiting dilution cloning or ClonePix FL. For advice regarding ClonePix cloning, see recommendations in the Freedom DG44 Kit manual or request a consultation at pd.direct@lifetech.com

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In the Freedom DG44 Kit, both Geneticin and DHFR selection are used, either sequentially or simultaneously.

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Detailed information about the pcDNA 3.3 TOPO TA vector and pOptiVEC TOPO TA vector can be found in the Freedom DG44 Kit manual.

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A certificate of analysis (COA) demonstrating suitability for research use is available to everyone who purchases the CHO DG44 Cells (cGMP banked) and Media Kit or the Freedom DG44 Kit. A full documentation package, including the cell line's lineage, history, and cGMP testing results, is offered in conjunction with a commercial use license

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All major components needed for the development of stable clones are included in the Freedom DG44 Kit: CHO DG44 Cells (cGMP banked), vectors for cloning one or two genes (pcDNA 3.3 TOPO TA Vector Kit and pOptiVEC TOPO TA Vector Kit), transfection regent ( FreeStyle MAX Reagent), competent cells (One Shot TOP10 Chemically Competent E. coli), serum-free cell culture media (CD DG44 Medium, CD OptiCHO Medium, and OptiPRO SFM), and other reagents such as Pluronic and Gentamicin. A complete list of components, including additional materials required but not included, can be found in the Freedom DG44 Kit manual.

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Yes. CHO DG44 Cells (cGMP banked) are intended (with the purchase of a commercial license) for the development of biologics. They have been produced in a cGMP animal origin-free environment, and have been scaled up to produce products for clinical testing.

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The stability of expression was assessed for over 60 generations with the observation that most clones maintain their level of expression after 60 generations. Expression levels were considered stable if the titer at the end of the stability assessment (at generation 60) was at least 70% of the titer observed at the beginning of the stability study.

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Product quality depends on multiple development aspects, such as the product itself, the selected clone, and the upstream and downstream process. CHO DG44 Cells (cGMP banked) offer all the typical CHO cell line benefits with respect to low protease levels and preferred glycan pattern.

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It is possible to go from transfection to lead clone in less than 6 months with the Freedom DG44 Kit.

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IgG titers of ≥0.5 g/L in simple fed batch and ≥1 g/L in fed batch with complex feeds have been obtained using the Freedom DG44 Kit. Titers of 20-100 mg/L with blood factors and other hard to express proteins (HTEP) have also been achieved with this kit.

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