During the past decade, biopharmaceutical manufacturers have witnessed significant productivity gains that can largely be attributed to developments in cell culture media design. However, a more holistic approach to process development is now emerging with the understanding that a quality-driven upstream process translates downstream to a high-quality final product.
Today, a primary aim of biologic manufacturers is to streamline the entire production process to deliver efficient and robust processes that are predictable and deliver a consistent product. Cell nutrition firms are now focusing on specific applications and process requirements utilizing platform media, feeds, and supplements that deliver value through performance and quality improvements.
This tutorial describes how Kerry supports process development scientists and biologic manufacturers in the optimization of their upstream process through the use of specifically designed cell culture supplements and feeds. In particular, the article describes Kerry’s yeast-derived supplement, UltraPep™ YE, a defined and consistent yeast extract that enhances CHO cell performance.
The paper also examines the application of Sheffield brand cell-line specific supplements, the Sheff-CHO series, and the advantages this strategy can deliver.
Enhancing CHO Cell Performance
Plant-based hydrolysates are an effective replacement for serum and animal-derived supplements in cell culture media. As non-animal-derived supplements, plant-based hydrolysates can deliver consistent benefits to cell performance parameters across a variety of cell lines and types. Although the manufacture of these supplements is now well-controlled, it is the complex nature of hydrolysates that has concerned process development scientists.
Addressing the concerns of hydrolysate complexity, Kerry has developed a new hydrolysate/yeast extract platform, called UltraPep. UltraPep YE is an animal-free, ultrafiltered yeast extract, designed for minimal variability. The highly optimized production process involves a nonanimal enzyme cocktail of both proteases and non-proteolytic enzymes. Designed with fewer processing steps and to specifically expand the range of nutritional factors available to the cell, UltraPep provides growth-promoting peptides as well as essential amino acids, carbohydrates, lipids, vitamins, and minerals.
Recent experiments compared CHO cells cultured in commercially available chemically defined media (CDM) alone to CDM supplemented with either UltraPep YE or another commercially available ultrafiltered yeast hydrolysate (UFYE). Cells were seeded at 4.0 x 105 cells/mL and grown in 125 mL shake flask to a final volume of 35 mL CDM (+1 mg/mL G-418) either alone with UltraPep YE or with the competitor UFYE (1 g/L). 1 mL samples were taken at day 0, 5, 7, 8, 9 to assess culture viability using a Nova BioProfile Flex automated analyzer. At day 12 product titer was analyzed via HPLC.
The results demonstrated significant performance benefits for the cultures supplemented with UltraPep YE. Peak cell density was achieved earlier in the growth cycle and was maintained for longer periods in cultures supplemented with UltraPep YE when compared to the CDM and UFYE control. This effect on cell growth translated to a significant increase in product titer with a threefold increase observed for the UFYE control and a greater than sixfold increase in titer for the UltraPep YE when compared to CDM alone (Figure 1A, Figure 1B).
Improving Cell Performance
As the nutritional requirements of individual production cell lines and clones becomes better understood, their response to culture media and supplements must be examined on a case-by-case basis to identify optimized solutions. For example, recent studies by Kerry have shown UltraPep YE and recombinant human serum albumin (rHSA), when added in combination, can have a positive effect on growth and productivity compared to the components alone.
Interaction of media components and the importance of optimizing supplement and feed addition according to the basal or platform media employed were investigated further. A single CHO clone was tested in two independent CDM (CDM-A and CDM-B) supplemented with a range of concentrations of UltraPep YE and the optimized dosage of UltraPep YE and rHSA. Experimental set up was as described for Figure 1 with regular samples analyzed for cell viability and total and specific productivity.
In CDM-A, CHO cell growth decreased with increasing hydrolysate concentration while product titer and specific productivity (Qp) increased. The optimized combination of UltraPep YE and rHSA demonstrated the greatest overall performance benefit (Figure 2A and 2B).
In contrast, cell growth and productivity was noticeably improved with respect to the CDM-C control when the medium was supplemented with 1 or 2 g/L UltraPep YE. As with CDM-A, CDM-C supplemented with 4 g/L UltraPep YE inhibited cell growth but increased product titer and Qp while the optimized dosage of UltraPep YE and HSA increased cell growth, translating to an increase in productivity (Figures 2C and 2D).
ligned with customer requirements and the trend toward QbD, Kerry developed cell-line specific complex media supplements. Proprietary process technology and innovative media optimization methods have been applied to develop specific, homogeneous, functional supplements from combinations of complex and chemically defined animal-component free media additives.
As an example, the Sheff-CHO system is a series of complete medium supplements optimized for CHO cells. The systems contain mixtures of factors known to promote CHO cell growth, cell metabolism, and protein production and have been carefully optimized to give superior recombinant protein yields.
The advantage of applying cell line specific supplements such as Sheff-CHO Plus ACF and Sheff-Pulse both in batch and fed batch cultures was recently investigated. Cells were seeded at 4.0 x 105 cells/mL and grown in 125 mL shake flask to a final volume of 50 mL CDM or DMEM plus Sheff-CHO Plus ACF at 9 g/L for batch cultures. For fed batch cultures media was supplemented at day 5, 7, 9, and 12 with basal media or a proprietary blend of Sheff-CHO Plus ACF and Sheff-Pulse. 1 mL samples were taken at day 0, 5, 7, 8, 9 to assess culture viability using a Nova BioProfile Flex automated analyzer. At day 12 product titer was analyzed via HPLC.
A similar study was set up to compare the Sheff-Pulse system with competitor feeds. Experimental design was as described above with cultures fed at day 5, 7, 9, and 12 and samples taken throughout the culture process to assess cell performance with respect to growth and productivity.
The addition of Sheff-CHO to commercially available CDM or the basal media DMEM was shown to significantly improve CHO cell culture performance when compared to cells grown in medium alone. This growth improvement was further enhanced with the addition of the feed (Figure 3A). The increased growth rate translated to a dramatic increase in product titer, with a greater than fourfold increase when the supplement was introduced as a feed (Figure 3B).
In the comparative study with competitors’ feed systems, Sheff-Pulse was seen to outperform with respect to both CHO cell growth and productivity in either CDM or DMEM (Figures 3C and 3D).
Media development strategies are a constant area of focus with simplification, predictability, and consistency of upstream processes as major goals. Increasingly, drug manufacturers are looking to raw material suppliers to provide quality products that are designed for purpose. Developments like UltraPep YE and Sheff-CHO systems demonstrate that incorporation of thoughtfully designed supplements can deliver value to manufacturers, leading to enhancements in cell performance, expediting process development, and reducing time to market.