The Role of Media Development in Process Optimization: An Historical Perspective

Abstract

Culture media development continues to be an area that offers the potential to dramatically improve the productivity of biopharmaceutical manufacturing processes. Having a well-defined media formulation optimized for maximum protein production can significantly improve product titers, thereby reducing costs and improving efficiency. In this article, we discuss the historical aspects of media development from the initial dominance of serum as a supplement in cell-culture media to the current trend toward serum-free, chemically defined, protein-free media customized for particular cell lines. We will also examine the advantages of developing a robust serum-free cell-culture medium containing defined animal-free protein supplements.

The past three decades have seen major developments in cell culture technologies in response to the increasing demand for approved biopharmaceuticals. Various approaches have been used to generate cell culture processes with the desirable traits of high titer, robustness, and improved scale-up efficiency. Key focus areas for upstream process development and optimization include choice of cell line and clone, expression vector design, medium optimization, and bioreactor conditions. This article examines approaches to media development in the biopharmaceutical industry over time, the impetus for change, and directions for the future.

Novozymes Biopharma

Initial Media Development

The first application of cell culture technology was for vaccine production in the 1950s. Traditionally, embryonated chicken eggs were used for vaccine production. Because of increased demand for vaccines, there was a need for new technologies that could produce large quantities. The first mammalian cells used to generate a commercial product were monkey kidney cells, which were used to produce the polio vaccine.¹ These were an attached cell line and required serum for growth. Further advances occurred during this time with the commercial production of an inactivated foot-and-mouth disease vaccine in baby hamster kidney (BHK) cells. This cell line was cultured in Eagle's medium, supplemented with 5% adult bovine serum up to a scale of 5,000 L.² Eagle's minimal essential medium, the first basal synthetic medium, contained a mixture of amino acids, vitamins, cofactors, carbohydrates, and salts to support cell growth. However, supplementation was also required, generally by addition of animal serum. Another major advancement in the use of mammalian cell culture for the production of biologics for human use came with the US Food and Drug Administration's acceptance of the use of continuous cell lines. Continuous cells lines such as Chinese hamster ovary (CHO) and myeloma cells (NSO and SP2/0) meant cells could be cultured in suspension and were readily scalable, allowing for increased product yields.

Serum as a Supplement

Bovine serum has been the supplement of choice during the development of general cell culture. Serum has been shown to provide all the essential nutrients for cell growth and productivity, including macromolecular proteins, low-molecular-weight nutrients, anti-oxidants, and carrier proteins for water-insoluble components, and is known to contain anti-apoptotic factors.³ Serum also contains a high concentration of albumin, which can protect cells from stress factors generated under bioreactor conditions such as pH nutrient fluctuations and shear forces, as well as a number of transport functions.⁴ Although serum delivered many benefits for the industry, serum also has important drawbacks, such as batch-to-batch variability, leading to a lack of process and product consistency, complex downstream processing to remove serum contaminants from the final product, and fluctuating costs. Even more important, the transmissible spongiform encephalopathies (TSEs) and other adventitious agents in bovine serum resulted in a strong regulatory drive for the biopharmaceutical industry to eliminate serum and other animal-derived components from the manufacturing process.⁵ A 1999 report by Wessman and Levings indicated that as much as 20–50% of commercial fetal bovine serum was virus-positive.⁶ These regulatory constraints led to the development of defined media supplements and the industry's move away from the use of serum to a serum-free medium.

Serum-Free Medium

Initially, serum-free medium was introduced into biopharmaceutical manufacturing because of the cost and process constraints of serum. Serum-free medium (SFM) was first used in mammalian cell culture by Ham in 1965. Much of this early work involved anchorage-dependent cell lines.^7–9 To achieve growth-promoting effects similar to those of serum, SFM often was supplemented with animal-derived components that replaced the role of serum, such as transferrin, albumin, insulin, and other biological extracts. Serum supplementation continues to be used in many early stage research applications, clonal development, and vaccine production.

Establishing more defined media regimes reduced the problems associated with the batch-to-batch variability seen with serum and allowed more consistent product and process control with simpler purification and downstream processing strategies. However, having more defined media often resulted in extended cell adaption times, reduced growth rates, and decreased product titers, all of which increased the costs of manufacture.

The most common serum-free supplements incorporated into a basal medium have been growth factor sources, transferrin, hydrolysates, and albumin. Even today, some of these supplements are animal-derived (transferrin, albumin), contain animal components, or are ill defined and are still used in manufacturing processes because of the lack of safe alternatives. The regulatory issues of possible contamination of the final product with adventitious agents and ill-defined processes therefore remain. Chemically defined SFM are commercially available, but for some cell lines it has not been possible to design a robust, animal-free, and chemically defined medium that will perform as well as serum. Often, different cell lines and clones exhibit a large degree of variability in their nutritional requirements to achieve optimal growth and performance, resulting in a lengthy and costly media development program.

Medium Supplements

The remainder of this article will discuss four media supplements commonly used in the biopharmaceutical industry and recent advances in their development.

Hydrolysates

Regulatory agencies have encouraged the development of serum-free media devoid of any animal-derived components to avoid the risk of introducing adventitious agents such as viruses and prions. Protein hydrolysates have been shown to have beneficial effects on cell growth and productivity and are a relatively effective alternative to the use of serum. Peptones derived from bovine milk or animal tissues, such as Primatone RL (MP Biomedicals, Irvine, CA), are capable of supporting a number of different cell lines in the absence of serum.¹⁰ However, non-animal hydroly-sates from microorganisms such as yeast¹¹ and plants including soy and rapeseed^12,13 are being investigated as supplements for supplying the nutritional requirements of mammalian cells in culture.

Although plant-based hydrolysates have been shown to promote growth and productivity,¹⁴ the industry has not fully embraced hydrolysates as a serum substitute. Primarily this has been because of a lack of chemical definition and lot-to-lot variability leading to process and product inconsistencies. Currently, manufacturers of hydrolysate supplements are addressing these concerns through novel enzymatic techniques and more refined processing to produce a more consistent product.¹⁵

Albumin

Albumin, in the form of bovine or serum albumin (BSA, HSA) is commonly used in cell culture media formulations for nutrient transport. SFM is frequently supplemented with serum albumin as a carrier for fatty acids, lipids, amino acids, and trace elements. Additional advantages of albumin as a cell culture supplement include its ability to bind toxic components present in culture and protect against mechanical damage such as shear stress in agitated cell culture systems. The successful replacement of BSA or HSA in SFM with recombinant forms of albumin or synthetic compounds such as pluronic has been achieved, however, requirements for albumin vary depending on the cell line. For example, the myeloma cell line, NSO, lacks the functional pathway for cholesterol synthesis and therefore requires cholesterol. Albumin has been used as a carrier of cholesterol, although cyclodextrins have been used as alternative carriers of cholesterol and other lipids in culture media.

In recent years, a variety of recombinant animal-free forms of albumin (rHA) have become commercially available. Up and coming areas requiring commercial cell culture processes, such as advanced tissue and stem cell therapies and regenerative medicine, are areas where rHA is likely to prove a compliant and consistent alternative to current albumin sources.¹⁶ Recombinant albumin, such as CellPrime rAlbumin AF-S (Millipore, Billerica, MA), and Recombumin (Novozymes, Bagsvaerd, Denmark), produced in Saccharomyces cerevisiae has been shown to be structurally identical to native albumin. Safety, tolerability, pharmacokinetics, and pharmacodynamics also have been studied, showing equivalence to native human albumin.¹⁷

Growth Supplements

Although cells may be adapted to grow in media devoid of growth factors, growth factor supplements still are essential for the growth of many cells in culture. Insulin traditionally has been used as a mitogen and also is involved in glucose amino acid uptake, lipid metabolism, and DNA synthesis.¹⁸ Recombinant insulin has been available since 1982 (Genentech, Eli Lilly) and is the most universal supplement in SFM. Although insulin is the growth factor of choice, it is required at supra-physiological concentrations (2–10 mg/mL) to support cell growth and viability under culture conditions.^19,20 It is widely accepted that insulin action is primarily through the activation of the IGF-I receptor (IGF-IR) rather than its own insulin receptor (IR).²¹

An insulin-like growth factor analog, LONG R³ IGF-I (Novozymes) has been developed that acts directly at a much higher potency on the IGF-IR and has been shown to be equivalent to or out perform insulin and IGF-I in supporting CHO cell growth and productivity.^22,23 LONG R³ IGF-I, an animal-free recombinant supplement typically is used at 200-fold lower concentrations than insulin because of its increased IGF-IR affinity. It has a distinct biological advantage over other growth factor supplements because of a 100-fold reduced affinity for inhibitory IGF-binding proteins. Stimulation of the IGF-IR results in the activation of a number of signalling pathways, some of which are known to have key mitogenic and anti-apoptotic effects. LONG R³ IGF-I results in greater activation of these signalling molecules, thereby increasing culture longevity and productivity. LONG R³ IGF-I has been shown to be an effective alternative to insulin as a growth factor supplement for sustaining cell growth and viability in serum-free culture at industrial scale.²⁴

Transferrin

Transferrin is required to transport iron into cells, which is essential for cell growth and the regulation of key metabolic processes, such as DNA synthesis and oxygen transport.²⁵ Transferrin has also been shown to play an important role in binding heavy metals in culture. Industrial cell lines such as CHO and NS0 require transferrin to attain optimal cell growth and productivity. Transferrin has been available in the form of serum-derived purified human transferrin (hTf) or bovine transferrin (bTf). Alternatively, inorganic iron salts have been used to supply iron to mammalian cells. To supply high-density cell cultures with sufficient iron, elevated concentrations of iron salts are required that use low affinity non-transferrin receptor pathways. This can have a negative effect on cell growth because of the formation of free radicals and oxidative stress from the unbound ferric or ferrous irons. Precipitation of iron hydroxide in the culture medium also can lead to limited bioavailability of iron to the cell.²⁶

Attempts to provide an efficient supply of iron by chemical chelators such as aurintricarboxylic acid or 2-hydroxy-2,4,6-cycloheptatrein-1-one (tropolone) has had limited application across a variety of cell lines because of unpredictability in controlling the intracellular redox cycle and cell oxidation processes.

A recombinant analogue of human transferrin, CellPrime rTransferrin AF (rTransferrin), (Novozymes) has shown equivalence to hTf and superiority to bTf in stimulating cell growth and productivity across a number of cell lines (Figure 1).^27,28 rTransferrin binds specifically to the transferrin receptor, facilitating iron uptake into the cell.

Figure 1

Supplements in Combination

The demand for a universal and robust cell culture media has led to the need to identify cell culture components that substitute for the growth-promoting effects of serum. Nutrient requirements for individual cell lines have been found to differ considerably and, therefore, it has been difficult to design a single serum-free media for the growth of cell lines of commercial interest. In response to this demand, combinations of essential serum proteins have been examined for their ability to stimulate cell growth and productivity in a variety of industrially relevant cell lines.

Studies in CHO cells have shown that growth and viability were adequately maintained in the absence of serum only when both IGF-I and transferrin were overexpressed in genetically engineered CHO cells.²⁹ The combined action of two recombinant forms of these serum proteins, IGF-I (LONG R³ IGF-I) and transferrin (CellPrime rTransferrin AF) on CHO cell growth and productivity in SFM has been investigated (Figure 2). Results from this study show that a combination of these two recombinant proteins promote a synergistic increase in the levels of cell growth and productivity above those obtained from a standard SFM or each protein on its own.

Figure 2

Conclusion

Biopharmaceutical companies require cell-culture media to be animal free, serum-free, defined, and cost-effective. In addition, there is a need to maintain or enhance process productivity while satisfying regulatory requirements for the elimination of serum components. A major driver for this has been the concern over contamination of the final drug product with adventitious agents derived from animal components. The industry has seen various attempts to produce robust animal-free, chemically defined media, and more recently, a protein-free media that is acceptable to regulatory agencies. However, the time involved in adapting cells to defined media, which often have resulted in reduced growth rates and product titers, has shown media development to be an important factor in the increasing cost of manufacture of the final drug product.

With a variety of recombinant, animal-free, defined protein supplements such as growth factors, transferrin, and albumin entering the market, the biopharmaceutical industry now has innovative and safer alternatives to serum and other animal-derived supplements. This situation allows manufacturers to rethink their media development to achieve greater process performance in a more regulatory compliant way.

Sally Grosvenor is a senior scientist and scientific communications manager at Novozymes, Ltd., Thebarton, Australia, +61 883547787, slyg@novozymes.com

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