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Mammalian cell expression systems are the dominant tool today for the production of complex biotherapeutic proteins. Different CHO hosts are used routinely to develop such biologics and are combined with several expression system like the dhfr, the UCOE or the glutamine synthetase (GS) system.

Cellvento® 4CHO medium and its companion Cellvento® 4Feed were developed to grow a broad range of CHO cells. The production medium supports initial cell growth and production while the highly concentrated, neutral pH feed is added to replenish depleted nutrients required for cellular function and to maintain and extend the production phase in fed-batch mode. This single feed is concentrated to more than 130 g/L allowing a reduction of the volume of feed added to the medium thereby increasing the volumetric productivity. It also contains cysteine and tyrosine derivatives which have been shown to release free cysteine and free tyrosine slowly throughout the culture. This time dependent release avoids tyrosine depletion which can lead to sequence variants. This process also results in free cysteine release, while maintaining a reduced redox environment. This is often related to higher cell growth and productivity.

As the performance of production media and their companion feed(s) are typically interdependent, optimizing a feeding strategy is a crucial step to achieve high cellular growth while maintaining high specific productivity. This document provides the basis for initiating feed optimization activities, but fine-tuning an effective feeding strategy should be considered.

 

The fed-batch media system

Cellvento® 4CHO medium and its companion Cellvento® 4Feed supplement are chemically-defined, non-animal origin products designed for use with CHO cell-based mammalian cell culture. The medium and its feeds are effective at achieving high-density cell growth and increased productivity with CHO suspension cell types. As with all fed-batch processes, optimization of feeding volumes and feed frequency are recommended.

 

Applications

• Cellvento® 4CHO medium and its companion feed have been designed for use with recombinant CHO suspension cells but may be suitable for other cell lines.
• The medium does not contain hypoxanthine and thymidine to allow a broad utilization, also in dhfrtransfected cells. The feed does not contain glucose to allow a fine tuning of the glucose concentration during fed-batch processes, thus allowing to minimize lactate production. The feed contains sources of both cysteine and tyrosine and should not be supplemented with any additional alkaline feed.
• Cellvento® 4CHO medium should be used as an amplification medium and a production medium in fed-batch applications.

 

Recommended feeding strategy

Cellvento® 4CHO medium and companion feed have been developed to complement each other and enhance the performance of CHO cells in protein production. As with most upstream bioprocesses, optimization of feed volumes and timing of feed administration should be empirically determined on a process- and cell-line specific basis to maximize performance. The table below provides recommended ranges for evaluation, of both feed volumes and frequency of feeding, to optimize each parameter within the context of an overall feeding scheme.

 

Fed-batch performance in spin tubes

Cell culture performance tests have been evaluated.

Cell growth and protein production profiles were virtually indistinguishable in the 3 production lots tested, ensuring that Cellvento® 4CHO medium can be used confidently with minimal risk of process variability attributable to cell culture media raw materials.

MilliporeSigma_ProcessGuidanceGraph1
Figure 1: Growth profiles in fed-batch culture. Cells from a CHOK1 GS were grown in Cellvento® 4CHO medium supplemented with complementary Cellvento® 4Feed product.

 

MilliporeSigma_ProcessGuidanceGraph2
Figure 2: Consistent IgG titers were achieved using three different lots of Cellvento® 4CHO medium against three different lots of Cellvento® 4Feed over a 14-day fed-batch culture with cells from CHO-K1 clone 1.

 

 

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