The transition from transient to stable production workflow with Chinese hamster ovary (CHO) cells requires a change in culture medium. On 22 August 2018, Shreya Lowmaster (field applications scientist at Thermo Fisher Scientific) discussed transient to stable solutions for moving from research to commercial production.

Lowmaster’s Presentation 
Researchers are demanding higher titers in transient systems, and many people use CHO systems as an alternative to HEK293 cells. The Thermo Fisher Gibco ExpiCHO Expression System kit was developed for transient expressing pools used in research. Now the company introduces Gibco ExpiCHO Stable Production Medium for commercial-scale production. These two products are integrated into one system.

In the transition, users typically need to screen and optimize media. Normally, a medium would be optimized for the cloning process and then adapted for manufacturing. It can take over three months to go through that process. With Gibco ExpiCHO Stable Production Medium, you can bypass this step and eliminate screening and optimization to save time.

In a transient-expression workflow, you begin transfection with your vector of choice and a transfection reagent along with Gibco ExpiCHO Expression Medium. After adding an enhancer and feed, then applying a temperature shift, you would harvest on day 14 depending on the protocol. If you need more product, you must repeat the entire workflow.

In a stable-expression workflow, using the same vector and transfection reagent paired with Gibco ExpiCHO Expression Medium, stable transfection is followed by two selection phases to identify top clones. A master cell bank of the final clone is cryopreserved to be used for multiple productions, saving time in the long run. After clone selection, you can switch to the Gibco ExpiCHO Stable Production Medium for scaling up.

ExpiCHO products can be used for both transient and stable production systems. For transient expression, ExpiCHO-S cells are high producers selected for high transfection efficiency. Gibco ExpiCHO Expression Medium is optimized to work with those cells and Gibco ExpiFectamine CHO Transfection kits for high transfection efficiency and titers. For stable production, you can use CGMP-banked ExpiCHO-S cells, Gibco ExpiCHO Expression Medium, and a Gibco ExpiFectamine CHO Transfection kit. You can switch to the commercial-scale medium (Gibco ExpiCHO Stable Production Medium) at any point after selection. It comes in both liquid and Advanced Granulation Technology (AGT) powder formats.

Case Study: After stable clone selection (with screening and stability assessment), Thermo Fisher Scientific had a stable ExpiCHO-S clone producing >3 g/L IgG for multiple adaptation experiments. We compared ExpiCHO Expression and Stable Production media with two competitors’ media, each paired with their respective feed. Cultures were passaged every three to four days in shake flasks and seeded with 200,000 cells/mL. The cells were thawed directly into Gibco ExpiCHO Stable Production Medium, requiring no adaptation. Even after five passages, cells grown in competitor media lagged in viable cell density (VCD).

In a small-scale bioreactor experiment, using liquid and AGT formats of ExpiCHO Stable Production Medium along with the recommended Thermo Fisher EfficientFeed C+ feed, comparable VCD results were shown between the two formats. In the same experiment, cells grown with Thermo Fisher products showed superior titers over those cultured in competitors’ media and feeds. The Thermo Fisher products also scaled up efficiently in a 50-L HyPerforma Single-Use Bioreactor (S.U.B.) with minimal process or feed optimization, producing 3.5 g/L on day 18.

Questions and Answers 
What is the difference between the Freedom CHO-S and ExpiCHO systems? The former is not part of a platform. The ExpiCHO platform facilitates research to manufacturing needs.

What vector do you recommend using with this platform? We recommend a vector (e.g., an antibiotic-based vector such as pcDNA 3.3 or 3.4) that requires minimal cell line engineering. Cytovance has worked with a different vector.

In the case study, what were the stability criteria, and what percentage of clones evaluated met them? The criterion was stability for 30 generations, with 20% of clones meeting that.

When do you switch from Gibco ExpiCHO Expression Medium to Gibco ExpiCHO Stable Production Medium? ExpiCHO Expression Medium is a transfection medium and ExpiCHO Stable Production Medium is a scale up medium. We recommend that you switch into the scale up medium after clone selection. ExpiCHO Stable Production Medium is not optimized to support high transfection efficiency.

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