Gibco™ Freedom™ DG44 Kit

Catalog No.	Quantity
A1373701	1 kit

Catalog No. A1373701 Supplier Gibco™ Supplier No. A1373701

Description

Freedom™ DG44 Kit

Gibco™ Freedom™ DG44 Kit is an easy-to-use, beginning-to-end product, for cloning and expression of recombinant proteins in dihydrofolate reductase deficient (DHFR-) Chinese Hamster Ovary cells. The Gibco™ Freedom™ DG44 Kit includes components for transfection, expression, clone creation, and stable cell line selection, all in a conveniently packaged kit. The Gibco™ Freedom™ DG44 Kit also delivers a proven host cell line with simplified commercial licensing options.

• Flexible commercial licensing without the need for royalties
• Proven host used in approved products and a wide array of proteins
• Complete workflow that can take your gene of interest from transfection to lead clone typically in less than 6 months
• Convenient packaging, for simplified ordering and storage
  
  Commercial Licensing that is Simple, Flexible and Without Royalties
  Multiple companies provide CHO (Chinese Hamster Ovary) stable cell line development services and products that require annual fees during development of your therapeutic protein. In addition, royalties may be required on the sales of your therapeutic protein upon commercialization. Gibco™ Freedom™ Kits provide you with the tools that enable you or your CRO provider to develop your stable cell line for just the price of the kit itself. Furthermore, as you move towards commercial use, a flexible license structure is available without the burden of royalties and works to keep it as simple as a onetime payment (Fig 1). For more information regarding licensing, email us at outlicensing@lifetech.com.
  
  Host Cell Background with Proven Results
  DG44 cells have been a choice of scientists for decades for the expression of IgG and numerous blood proteins. Protein expression levels can vary significantly depending on the class of protein, however gram per liter levels of IgG expression has been demonstrated using these DG44 parental cells. So you can be confident that if your work calls for use of DG44 cells, they will deliver expected protein levels.
  
  Complete, Fast, and Efficient Workflow
  The Gibco™ Freedom™ DG44 Kit’s complete, proven, and fully integrated workflow allows you to move from transfection to lead clone typically in less then 6 months, helping to reduce hands on time and Fulltime Equivalent (FTE) requirements (Fig 2). We have implemented a single round of MTX (methotrexate) amplification in the protocol, which in our hands provided up to 120 fold titer improvement in less than 6 weeks (Fig 3). The entire workflow is performed in animal origin free (AOF) medium. The kit also offers the following advantages:
   
• Cloning of one or two genes that encode your protein(s) of interest
• High efficiency transfection of DNA into DG44 Cells (cGMP-banked)
• Included flash drive with loaded manual
  
  Access to Gibco™ Freedom™ Kit Support
  If any specific support is needed beyond the manual, please contact your account manager to obtain a Process Science Manager consultation. In addition, one of our support scientists may check in with you to ensure that your experience with the kit is as successful as possible. For further assistance, please email our support team at GibcoServices@thermofisher.com.

Specifications

• Content And Storage: CD DG44 Medium: 2°C to 8°C, protect from light
  CD OptiCHO™ Medium: 2°C to 8°C, protect from light
  OptiPRO™ SFM: 2°C to 8°C, protect from light
  FreeStyle™ MAX Reagent: 2°C to 8°C, do not freeze
  Pluronic™: F68 15°C to 30°C
  
  DG44 (CHO) cGMP Banked suspension dhfr (dihydrofolate reductase) Negative Cells: liquid nitrogen
  One Shot™ TOP10 Chemically Competent E. coli: –80°C
  pcDNA 3.3 TOPO™ TA Vector Kit: –5°C to –20°C
  pOptiVEC™ TOPO™ TA Vector Kit: –5°C to –20°C
  Gentamicin™: –5°C to –20°C
• Product Line: Freedom
• Quantity: 1 kit

Frequently Asked Questions (FAQs)

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.

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.

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).

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.

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.

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.

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).

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).

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.

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.

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.

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

Only two transfections are recommended.

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

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

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.

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.

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

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.

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.

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.

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.

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.

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.

For Research Use Only. Not for use in diagnostic procedures.