Freedom™ DG44 Kit - FAQs

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26 product FAQs found

在Freedom DG44试剂盒中，将蛋白亚基插入pcDNA3.3-TOPO TA载体或pOptiVEC-TOPO TA载体，会产生不同的后续效应吗？

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。

在Freedom DG44试剂盒中，由于MTX扩增仅对pOptiVEC TOPO-TA载体所表达的蛋白亚基起作用，我应如何确认pcDNA3.3-TOPO TA载体所表达的蛋白亚基也实现了扩增？

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Freedom DG44试剂盒中，pOptiVEC-TOPO TA载体包含了一个DHFR营养缺陷型筛选标志物，这样用户就可免除蛋白生产过程中的抗生素筛选步骤。不过，这对于包含新霉素筛选标志物的pCDNA3.3-TOPO TA载体而言是不可行的。能否在蛋白生产过程中加入G418成份？

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。

In the Freedom DG44 kit, does it matter which protein subunit is inserted into the pcDNA3.3-TOPO TA versus the pOptiVEC-TOPO TA vectors?

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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

In the Freedom DG44 kit, since MTX amplification would only work for the protein subunit expressed by the pOptiVEC TOPO-TA vector, how can I ensure that the protein subunit expressed by pcDNA3.3-TOPO TA is also being amplified?

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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

In the Freedom DG44 kit, the pOptiVEC-TOPO TA vector contains a DHFR auxotrophic selection marker so that one can avoid using antibiotic selection during protein production. However, this is not possible with the pcDNA3.3-TOPO TA vector as it contains a neomycin selection marker. Is it okay to have G418 present during protein production?

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).

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Can MTX be used at concentrations higher than 4 µM to increase protein yield using the OptiCHO Express Kit?

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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

How can I learn more about commercial licensing of Freedom kits, ExpiCHO-S Cells (cGMP banked), CHO-S Cells (cGMP banked), and CHO DG44 Cells (cGMP banked)?

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

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Do you offer technical support for the Freedom DG44 Kit?

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

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

How can I learn more about commercial licensing of CHO DG44 Cells (cGMP banked)?

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).

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Is there a pull-through royalty on final products or an annual maintenance fee when using the Freedom DG44 Kit??

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).

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

When is a license required for the Freedom DG44 Kit?

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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

What are some considerations for successful scale-up of CHO DG44 Cells (cGMP banked)?

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

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

What are some considerations for obtaining a high number of stable clones using the Freedom DG44 Kit?

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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

How many transfections are typically required to get high-expressing clones from the Freedom DG44 Kit?

Only two transfections are recommended.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

What transfection methods can be used with the Freedom DG44 Kit?

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

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

What cloning methods can be used with the Freedom DG44 Kit?

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

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

What selection system is used in the Freedom DG44 Kit?

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

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Where are the Freedom DG44 Kit vector maps located?

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

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Does the Freedom DG44 Kit contain documentation for the CHO DG44 Cells (cGMP banked)?

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

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

What is included in the Freedom DG44 Kit?

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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Can CHO DG44 Cells (cGMP banked) be used for the production of commercial products?

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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

How successful is the Freedom DG44 Kit in creating stable clones?

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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

What level of product quality is seen with the Freedom DG44 Kit?

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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

How long does it take to obtain a lead clone with the Freedom DG44 Kit?

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

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

What titers can be achieved with the Freedom DG44 Kit?

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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.