A New Era in Cell Culture Media Development

Optimized cell-culture media is essential to enhance production of a quality biopharmaceutical product. In the past decade, the biopharmaceutical industry has witnessed previously unimaginable productivity gains that can largely be attributed to improvements in cell-culture media.

Historically, media contained animal-derived serum to maintain optimal mammalian cell growth and productivity. The recent introduction of regulations requiring the removal of serum and other animal-derived components in all biomanufacturing process has led to the introduction of leaner, more defined animal-component-free media (ACF). This shift in media design is now almost complete. However, the biopharmaceutical industry is always looking for improvements to reduce process timelines and time to market. Where once process development scientists strived for the one-media-fits-all approach, a new paradigm is now emerging with media being optimized for a particular cell line, clone, or process.

The primary challenge for the biopharmaceutical industry today is streamlining of the entire production process to deliver efficient and robust processes that are predictable and deliver a consistent product. Media developers are now focusing on individual processes and product requirements with the development of platform media containing optimized feeds and supplements to meet their specific process needs. A holistic approach to process development is becoming more common, with the understanding that a quality optimized upstream process translates downstream to a quality final product.

This article examines different media-development strategies that allow process-development scientists and biologic manufacturers to optimize their upstream process through the use of specifically designed supplements and feeds. Specifically, the article describes how plant-based hydrolysates are a proven and effective alternative to serum- or animal-derived media supplements, how complex and chemically defined hydrolysates can enhance process performance in a cell-line specific way, as well as describing the development of more defined and consistent hydrolysate products that further acknowledge the process-specific trends in media development.

HYDROLYSATES AS SERUM REPLACEMENTS

Plant-based hydrolysates were one of the first nonanimal-derived supplements to replace serum and performed as a robust media across a variety of cell lines and types. Known to provide amino acids, peptides, carbohydrates, vitamins, and minerals, hydrolysate supplements have delivered growth rates and product titers comparable to serum or complex chemically defined media.

A recent study examined hydrolysate fractions to determine functionality. Interestingly, not only were hydrolysates found to be an important source of free amino acids but also appeared to stimulate more efficient cell metabolism. In addition, the oligonucleotide fraction was found to remain largely intact during culture conditions, suggesting a role other than as nutrition, such as a possible protective role from shear stress or anti-apoptotic function to promote enhanced growth and productivity.

This effect on cell metabolism has been observed for both hybridoma (Sp2/0) and Chinese hamster ovary cells (CHO) cultured in chemically defined media (CDM) supplemented with plant-based hydrolysates. A metabolic shift was observed where the cultured cells began to metabolize lactate as the carbon source once glucose and glutamine had almost been consumed. In addition, both glucose and glutamine were consumed at a more rapid rate in supplemented cultures.

Accumulation of lactate during mammalian cell culture is a major concern because it can adversely affect cell growth and product quality. The metabolic shift observed when CDM was supplemented with plant-based hydrolysates resulted in an extended growth period and significant increase in product titer (see Figure 1).

Figure 1: A metabolic shift occurred when Chinese hamster ovary (CHO) cells were cultured in chemically defined media plus plant-based hydrolysate (HyPep 1510), resulting in increased cell density and productivity. (ALL FIGURES ARE COURTESY OF THE AUTHOR)

NEW PLATFORM TO ADDRESS HYDROLYSATE DEFINITION AND VARIATION

Plant-derived protein hydrolysates have been shown to be an effective replacement for serum and animal-derived components, delivering consistent benefits to cell performance parameters. Although the manufacture of these products is well controlled, hydrolysates by their own nature are complex and not fully defined.

With the aim of streamlining biologic production processes and reducing costs, consistency and predictability are becoming a major focus for the biotech industries. Acknowledging these trends, a new hydrolysate platform has been developed, which maintains the animal-free requirements of the supplement while minimizing variability issues and delivering a more reproducible product.

The production process is based on an innovative approach to enzymatic digestion involving a non-animal enzyme cocktail of both proteases and nonproteolytic enzymes. The highly optimized process is designed to expand the range of nutritional factors available to the cell, providing growth-promoting peptides as well as essential amino acids, carbohydrates, lipids, minerals, and vitamins. Importantly, product consistency can be enhanced and interlot variability can be reduced because the process requires fewer process steps.

Experimental data obtained from the comparison of CHO cells cultured in CDM alone to CDM supplemented with ultrafiltered yeast extract (UFYE) demonstrated significant performance benefits in the supplemented cultures. Peak cell density was achieved earlier in culture and was maintained for longer for UFYE when compared with the CDM control (see Figure 2a). The effect on cell growth translated to a significant increase in product titer (see Figure 2b).

Figure 2: Chinese hamster ovary (CHO) cell cultures supplemented with yeast hydrolysate maintained higher cell densities compared with chemically defined medium (a) translating to an increase in product titer (b); YE is yeast extract; UFYE is ultrafiltered yeast hydrolysate.

PROCESS PERFORMANCE AND MEDIA COMPONENTS INTERACTION

The initial cause for the shift away from serum in cell-culture processes was because of its complexity and ill-defined composition. Manufacturers had to struggle with inconsistent process performance, which ultimately affected product quality.

The development of CDM containing complex component formulations has meant cell viability, cell growth, and good product titer can be maintained without the use of serum. CDM provides many benefits for the manufacture of biotherapeutics, not the least of which are regulatory compliance, simplified downstream processing, and consistency of both processes and products. Although CDM can be costly, these performance benefits are important in driving improved manufacturing processes and therefore drug-development costs down.

As the understanding for specific nutrient requirements for various production cell lines increases, media formulations are being developed to achieve optimal performance. To enhance culture performance, CDM is often supplemented with defined feeds and supplements at predetermined stages in the growth and productivity cycle. However, optimization of these strategies can be time-consuming and success of this strategy is often dependant on the basal medium formulation.

Hydrolysates, a common cell culture supplement and feed, share a number of components with CDM. The additional availability of these common media components to the cell can have an additive effect, stimulating cell growth and productivity. Alternatively, the additive effect may have a negative impact on cell performance as a result of unintentional overdosing of certain components.

To demonstrate the interaction of basal media components and supplements on biological systems, CHOK1 cells expressing a recombinant protein, were cultured in two independently sourced CDM at full strength and 80% strength, either alone or supplemented with plant-derived protein hydrolysate. The results showed supplementation of chemically defined media with plant-derived protein hydrolysates affected cell culture performance in a medium-dependent manner. Interestingly, supplementation with hydrolysate of diluted CDM achieved equivalent or enhanced cell culture performance compared with full strength supplemented CDM (see Figure 3).

Figure 3: Supplementing two commercial chemially defined media (CDM) at 100% and 80% strength with 8g/L cotton seed hydrolysate (CSH) resulted in enhanced Chinese hamster ovary (CHO) cell viability (a and c) and productivity (b and d).

COMBINATIONS OF SUPPLEMENTS AND CELL GROWTH

Cell culture media for the production of biologicals are becoming more complex. As described above, the addition of media feeds and supplements to enhance cell culture performance must be carefully matched with the basal media because of component interactions.

With the nutritional requirements for individual production cell lines and clones becoming better understood, the response to culture media and supplements must be examined in a case-by-case basis. With the implementation of design of experiment and high throughput screening of media components it is becoming increasingly possible to design and optimize supplements and feeding strategies that address the specific nutrient requirements for each cell line and clone.

Addition of certain supplements in combination has recently been shown to have a positive effect on specific cell growth and productivity. An example of cell culture supplement interaction having a positive effect of cell performance has been observed when ultrafiltered yeast extract and recombinant human serum albumin (rHSA) were co-supplemented in CHO CDM. The ultrafiltered yeast extract, a consistent animal-free hydrolysate, at 1 g/L in combination with rHSA 1 g/L were shown to have a positive effect on growth and productivity when added alone compared with the CDM control. Moreover, when these supplements were added in combination, a synergistic response was observed for both CHO cell growth and productivity (see Figure 4).

Figure 4: Addition of the yeast extract and human serum albumin (HSA) supplements both alone and in combination enhanced total Chinese hamster ovary (CHO) cell density, which significantly affected product titer.

Interaction of media components and the importance of optimizing supplement and feed addition according to the basal or platform media employed were further investigated. A single CHO clone was tested in two independent CDM supplemented with a range of concentrations of ultrafiltered yeast hydrolysate and an optimized dosage of ultrafiltered yeast hydrolysate (YE) and rHSA. In CDM–A, CHO cell growth decreased with increasing hydrolysate concentration while product titer and specific productivity (Qp) increased. The optimized combination of YE and rHSA demonstrated the greatest overall performance benefit (see Figures 5a and 5b). In contrast, addition of YE alone in CDM–B had little effect on the already poor cell growth. In the presence of the combination of YE and rHSA, however, CHO cell growth displayed rates comparable to that of CDM–A. The combination of supplements again delivered best overall performance with respect to CHO cell growth and productivity (see Figures 5c and 5d).

Figure 5: Chinese hamster ovary (CHO) cell response to supplementing with yeast extract (YE) was shown to be dependant on background media. In contrast, cell performance was consistently improved when supplementing with the optimized dosage of yeast extract (YE) and human serum albumin (HSA). CDM is chemically defined medium.

MEDIA DEVELOPMENT USING QBD PRINCIPLES

To predict the interaction of media components feeds and supplements and the effect they may have on cell performance, media and supplement suppliers must work closely with their customers to understand their specific process requirements.

Quality has become increasingly important at all stages of the biomanufacturing process, supported by the implementation of quality by design (QbD) concepts. With the emphasis on both product and process understanding, QbD is being applied at the product/process development stage. If these principles are to be applied to the complete biomanufacturing processes, then it must include raw materials such as media components, which as mentioned are prone to variability and are not directly controlled by process development scientists.

Availability of detailed information about raw-material quality specifications can vary among suppliers and is often dependant on customer relationships. Lack of raw material understanding or protection of formulation information can make it difficult to call some supplements truly defined. Some suppliers work closely with their customers to ensure that all aspects of product quality meet biomanufacturers' process requirements.

Cell-line specific complex media supplements developed by Sheffield Bio-Sciences are current examples of the supplier acknowledging the change in media design and listening to customer requirements. Created using innovative media optimization methods, the supplements are manufactured using proprietary process technology to enhance performance of various biopharmaceutical production systems. This process allows for the combination of complex and/or chemically defined animal-component free media additives into a single homogeneous functional supplement.

As an example, The Sheff–CHO system is a series of complete medium supplements optimized for Chinese hamster ovarian (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.

In recent studies, the application of cell line specific supplements such as Sheff–CHO ACF Plus and Sheff–feed both in batch and fed batch cultures was examined. The addition of Sheff–CHO to commercially available CDM or the basal media Dulbecco's Modified Eagle Medium (DMEM) was shown to significantly improve CHO cell culture performance when compared with cells grown in medium alone. Peak viable cell density was seen to be at least 2-fold greater for medium supplemented with Sheff–CHO. This growth improvement was further enhanced with the addition of the feed (see Figure 6a).The increased growth rate translated to increased product titer, with a greater than 4-fold increase when the supplement was introduced as a feed (see Figure 6b).

Figure 6: Addition of Sheff–CHO supplements commercially available chemically defined media (CDM) or basal media significantly improved CHO performance compared with medium alone. Feeding with Sheff–Feed further enhanced both Chinese hamster ovary (CHO) cell growth and productivity.

In a comparison with competitors' feed systems, the Sheff–feed was seen to outperform with respect to both CHO cell growth and productivity in either CDM or DMEM (see Figures 7a and 7b). Interestingly, while the competitor feeds had minimal effect on cell performance in the basal media, DMEM, the Sheff–feed enhanced overall cell performance equivalent to that in CDM.

Figure 7: Comparison of Sheff–Feed against two competitor feeds in either chemically defined media (CDM) or Dulbecco’s Modified Eagle Medium (DMEM) for Chinese hamster ovary (CHO) cell growth and productivity. Sheff–Feed enhanced CHO cell performance compared with both competitor feeds.

THE NEXT GENERATION IN MEDIA DESIGN

As mentioned earlier, optimizing the upstream manufacturing process in terms of quality, predictability and consistency is dependent on all process materials and affected by supplier and customer relationships.

The importance of these relationships and the desire for the supplier of media supplements to address customer needs has seen the development of specialized media optimization services. Such services allow suppliers to work in conjunction with media development scientists to gain a better understanding of how various supplements and feeds function in different cell culture systems.

Media optimization services allow confidential information to be shared between the supplier and customer under specified agreements. This arrangement provides transparent opportunities in media enhancement strategies such as chemically defined, animal-component free, and serum-free media (SFM) optimization for individual applications. Solutions can be quite creative and cost-effective when such collaborative approaches are adopted and long-term relationships are forged. In some cases, a perpetual service agreement is enacted to allow for continued technical support and service. This value-added service can result in dramatic advances in productivity and consistency.

For example, studies examining the benefit of adding various supplements in the Sheff-System to either a commercially available SFM or the basal medium, DMEM, demonstrated a significant effect on CHO cell productivity. Supplements were tested at both the low and high recommended concentrations. Increases in product titer were observed in all supplemented cultures with increases almost 5-fold greater for SFM supplemented with a chosen supplement (see Figure 8a) and up to 10-fold in the supplemented basal medium (see Figure 8b).

Figure 8: Low and high recommended concentrations of Sheff–CHO products were supplemented in two types of basal media, SFM4CHO(a) or DMEM(b), and total productivity (solid) and specific productivity (Qp, hashed) measured. Increases in product titer were observed in all supplemented cultures.

Close customer relationships also provide an opportunity for suppliers to recognize evolving requirements and concerns in process and product development, improve existing systems, and develop new solutions designed specifically for cell culture and vaccines clients.

CONCLUSION

In an attempt to lower production costs, and improve time to market while increasing product output, the biopharmaceutical industry is looking to streamline production processes. Media-development strategies are a constant area of focus with simplification, predictability, and consistency of upstream processes as major goals.

The response to industry requirements has seen major advancements in areas of cell culture media design with respect to supply of specifically designed supplements and feeds. In addition, trends towards incorporation of QbD principles across the entire manufacturing process has meant drug manufacturers are looking to raw material suppliers to provide quality products that are fit for purpose.

Media and supplement system suppliers are also moving towards specialized performance optimization services, allowing for the transfer of information freely between supplier and customer to develop specific media formulations. These custom-designed products and services can not only provide manufacturers with enhanced upstream process performance but also have the potential to reduce process timelines and time to market.

Sally Grosvenor is a freelance writer and science communicator.