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Cultivating cell culture.


The past three decades have seen major developments in cell culture technologies in response to increasing demand for approved biopharmaceuticals. Various approaches have been employed to generate cell culture processes with the desirable traits of high titre, robustness and improved scale-up efficiency. A well-defined media formulation optimized for maximum protein production can significantly improve product titres, thereby reducing costs and improving efficiency. In this article, the author discusses the historical aspects of media development and will also examine the advantages of developing a robust serum-free cell culture media containing defined animal-free protein supplements.

In the Beginning ...

Cell culture was first utilized in vaccine production in the 1950s, replacing the traditional method of using embryonated chicken eggs; the increasing demand for vaccines required new technologies that were able to produce large quantities of vaccine. The first mammalian cells used to generate a commercial product were monkey kidney cells, producing the polio vaccine; these were an attached cell line and required serum for growth. (1) Other major advances in biologics included the following:

* continuous cell lines such as Chinese hamster ovary (CHO) and myeloma cells (NSO and SP2/0)

* the commercial production of an inactivated Foot and Mouth Disease vaccine in baby hamster kidney (BHK) cells. (2)

Pros and Cons of a Serum Supplement

Bovine serum has been shown to provide all the essential nutrients for cell growth and productivity and, as a result, has been widely used as a supplement throughout the development of general cell culture. Bovine serum that contains macromolecular proteins, low molecular weight nutrients, antioxidants and carrier proteins for water-insoluble components is also known to contain antiapoptotic factors. (3) In addition, serum contains a high concentration of albumin that is believed to protect cells from the shear forces and stress factors that are generated under bioreactor conditions. (4) Albumin also plays a role in nutrient and fatty acid transport. Although serum delivers many benefits for the cell culture industry, there are also a number of drawbacks, such as complex downstream processing to remove serum contaminants from the final product, batch-to-batch variability leading to a lack of process and product consistency, and fluctuating costs that have led to a shift away from the use of serum to a serum-free medium.

In addition to these problems, a report by Wessman and Levings in 1999 indicated that as much as 20-50% of commercial foetal bovine serum was virus-positive with the transmissible spongiform encephalopathies (TSEs). (5) These 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. (6) Such regulatory constraints led to the development of defined media supplements and contributed to the industry's move towards the use of a serumfree medium.

Serum-Free Media

Serum-free media (SFM) was first used in mammalian cell culture by Ham in 1965; much of the early work involved anchorage-dependent cell lines. (7-9) SFM was often supplemented with animal-derived components that replaced the role of serum, such as transferrin, albumin, insulin and other biological extracts to utilize the growth-promoting effect of serum. Establishing more defined media regimes reduced the problems with the batch-to-batch variability associated with serum and allowed more consistent product and process control with simpler purification and downstream processing strategies. However, a more defined medium often resulted in extended cell adaption times, reduced growth rates and decreased product titres, all of which negatively impacted on manufacturing costs.


The most common serum-free supplements have been growth factor sources, transferrin, hydrolysates and albumin incorporated into a basal medium. Even today, some of these supplements contain animal components, are animal-derived (transferrin, albumin) or are ill-defined and are still in manufacturing processes because of a lack of safe alternatives. Therefore, the regulatory issues concerning contamination of the final product with adventitious agents, as well as ill-defined processes, remain. Commercially available chemically defined SFMs are offered, but for some cell lines it has not been possible to design a robust animal-free, chemically defined media that will perform as well as a serum substitute. Serum supplementation continues to be used in many early stage research applications, clonal development and vaccine production. 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 programme.

Popular Media Supplements

The remainder of this article will discuss recent advances in the development of four commonly used serum supplements that address many of the concerns of the cell culture industry.

Hydrolysates: The development of serumfree media has been strongly encouraged by the regulatory authorities to minimize the risk of infection from viruses and prions. Peptones derived from bovine milk or animal tissues, such as Primatone RL, are capable of supporting a number of different cell lines in the absence of serum. (10) However, non-animal hydrolysates from micro-organisms such as yeast and plants, including soy and rapeseed, [12, 13] are being investigated as supplements for supplying the nutritional requirements of mammalian cells in culture. (11-13) Protein hydrolysates are a relatively effective alternative to the use of serum as they have been shown to have beneficial effects on cell growth and productivity.

The industry has not fully embraced hydrolysates as a serum substitute, despite the fact that plant-based hydrolysates have been shown to have growth and productivity promoting attributes. (14) Primarily, this has been as a result of batch-to-batch variability and a lack of chemical definition. Currently, manufacturers of hydrolysate supplements are addressing these issues through more refined processing and novel enzymatic techniques to produce a more consistent product. (15)

Albumin: Bovine or human serum albumin (BSA, HSA) is commonly used for nutrient transport in cell culture media formulations. Serum albumin is frequently used to supplement SFM 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 the toxic components present in culture and protect against mechanical damage such as shear stress in agitated cell culture systems. BSA or HSA in SFM have been successfully replaced with recombinant forms of albumin or synthetic compounds such as pluronic because of safety considerations, lot-to-lot variability and cost. 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. Recently, a variety of recombinant animal free forms of albumin (rHA) have become commercially available. (16,17)

Transferrin: Industrial cell lines such as CHO and NSO require transferrin to attain optimal cell growth and productivity. It 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. (18) Transferrin has been available in the form of serum-derived purified human transferrin (hTf) or bovine transferrin (bTf). As an alternative to transferrin, inorganic iron salts have been used to supply iron to mammalian cells. Elevated concentrations of iron salts that utilize low affinity non-transferrin receptor pathways are required to supply high-density cell cultures with sufficient iron. This can result in oxidative stress and the formation of free radicals from the unbound ferric or ferrous ions and can consequently have a negative effect on cell growth.

The precipitation of iron hydroxide in the culture medium can also lead to the limited bioavailability of iron to the cell. (19) The 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. CellPrime rTransferrin AF (rTransferrin), expressed in Saccharomyces cerevisiae, is a recombinant analogue of human transferrin and has shown equivalence to hTf and superiority to bTf in stimulating cell growth and productivity across a number of cell lines (Figure 1). (20,21) Supplied as human holotransferrin, rTransferrin binds specifically to the transferrin receptor, facilitating iron uptake into the cell for maximal cell culture performance.


Growth Factor Supplements

Despite the fact that cells may be adapted to grow in media without the growth factors, they are essential for growth of many cells in culture. Insulin has traditionally been used as a mitogen and is also involved in glucose, amino acid uptake, lipid metabolism and DNA synthesis. (22) Recombinant insulin is the most universal supplement in SFM and has been available since 1982 (Genentech, Eli Lilly). Although insulin is the growth factor of choice, it is required at supraphysiological concentrations (2-10 mg/mL) to support cell growth and viability under culture conditions. (23,24) 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). (25) Development of an insulin-like growth factor analogue (LONG R3IGF-I) that acts directly, at a much higher potency, on the IGFIR has been shown to be equivalent to or outperform insulin and IGF-I in supporting CHO cell growth and productivity. (26,27)

Supplements in Combination Promote Cell Growth

In response to demand for a robust and universal serum-free cell culture media, cell culture components that substitute for the growth promoting effects of serum are being analysed. It has been difficult to design a single serum-free medium for the growth of all cell lines because nutrient requirements differ considerably between individual cell lines. As a result, 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 only when both IGF-I and transferrin were over-expressed in genetically engineered CHO cells, was growth and viability adequately maintained in the absence of serum. (28) The combined action of two recombinant forms of these serum proteins, IGF-I (LONG R3IGF-I) and transferrin (CellPrime rTransferrin AF) on cell growth and productivity of CHO cells in SFM has been investigated. Results from this study show that a combination of these two recombinant proteins promotes a synergistic increase in the level of cell growth and productivity above that obtained from a standard SFM or each protein on its own.


A major driver for the development of animalfree and serum-free cell culture media has been the concern regarding contamination of the final drug product with adventitious agents derived from the animal components. It is essential that new animal-free products maintain or enhance process productivity whilst satisfying regulatory requirements for the elimination of serum compounds.

The industry has seen various attempts to produce a robust, animal-free, chemically defined, cost-effective medium and, more recently, a protein-free medium that is acceptable to regulatory agencies. However, the time involved in adapting cells to defined media, often with reduced growth rates and product titre, has shown media development to be an important factor in elevating the cost of manufacture of the final drug product. The biopharmaceutical industry now has a choice of innovative protein supplements from a variety of recombinant, animal-free, defined products, such as growth factors, transferrin and albumin, which have recently entered the market. This situation allows manufacturers to achieve greater process performance using safe alternatives to animal-derived supplements and serum in a regulatory compliant way.

For more information

Sally Grosvenor is Scientific Communications

Manager at Novozymes Biopharma AU Ltd,

Adelaide, Australia.


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Title Annotation:cell culture
Author:Grosvenor, Sally
Article Type:Report
Geographic Code:8AUST
Date:Sep 1, 2008
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