Cultivation of mammalian cells has historically been performed using relatively ill-defined nutrient conditions (Mather 1998; Freshney 2000; Altman & Dittmer 1961). In an attempt to mimic the composition of bodily fluids, tissue explants and monodisperse cells were bathed in an iso-osmotic buffered saline solution augmented by addition of various organic nutrient constituents, and ultimately further supplemented by animal sera at 5-20% (v/v) (Jayme & Blackman 1985; Ham & McKeehan 1979; Barnes et al. 1984; Waymouth 1972).

In the decades following these initial cell culture efforts, there has been a dramatic evolution in the parameters and applications that define this field. As noted in Table 3.1, there has been a virtual explosion in the breadth of cell types, cultivation conditions and analytical tools to facilitate optimization and delivery of required nutrients. Concomitant with these trends has been an emergence of applications targeted toward production of biological molecules and engineered cell types for human and veterinary therapeutics that has evoked a rapid progression in the development of nutrient media to support these highly regulated applications (Jayme & Blackman 1985; Jayme & Gruber 1998).

About 15 years ago when I first lectured on development of serum-free media, I crafted a table that defined the various motivations to reduce or eliminate serum (Table 3.2). As a by-product of the cattle industry, animal serum (particularly foetal bovine serum, FBS) experienced extremes of supply and cost pressures that prompted both academic researchers and the emerging biotechnology industry to consider options for serum reduction or to qualify serum-free alternatives.

In addition to these concerns regarding price and availability, there was mounting evidence that serum addition might be problematic for certain cell culture applications (Ham & McKeehan 1979; Barnes et al. 1984; Waymouth 1972; Jayme & Gruber 1998). Serum factors typically promoted fibroblast overgrowth in mixed cell populations or failed to provide essential growth factors in adequate abundance to promote epithelial cell growth. Progenitor cells were difficult to maintain in serum-supplemented media without undergoing spontaneous differentiation or apoptosis. Differentiated cells exhibited rapid deterioration of key cellular functions. Serum also contained proteolytic enzymes that degraded cell-secreted products and neutralizing antibodies that reduced viral titres.

Finally, almost as an afterthought, I observed that animal serum might be a source of regulatory concerns, due to the antigenicity of foreign protein elements and the potential for them to introduce adventitious contaminants. At that time, little was known (Spier 1983) regarding viruses

Medicines from Animal Cell Culture Edited by G. Stacey and J. Davis © 2007 John Wiley & Sons, Ltd

Table 3.1 Evolution of cell culture applications.

Cell culture parameter

Historical requirement

Current requirement

Range of applicable cell types Serum supplementation Nutritional requirements Nutrient optimization method Inoculation density Maximal cell density Target culture application Regulatory level Range of bioreactor types Bioreactor controls

Narrow range

Relatively high

Less fastidious

Less analytical

Narrow range

105-106 cells/ml

Cell proliferation

Research/in vitro diagnostic

Narrow range

Limited manual control

Broad range

Relatively low or serum-free More fastidious More analytical Broad range 106-109 cells/ml Biological production Bioproduction/ex vivo therapy Broad range

Computer-driven controls that could pass transplacentally and that would be present even in aseptically collected foetal serum processed through multiple 0.1|im sterilizing filters. Prions were yet to become an issue for biological medicines and transmissible spongiform encephalopathies were not on anyone's radar screen in biotechnology.

Many investigators were reluctant to convert from their established serum-supplemented culture systems because of the anxiety in verifying comparability. When the serum supply and cost returned temporarily to previous levels, many laboratories deferred their prior efforts to develop serum-free media. Those who had ventured to eliminate serum discovered that early commercial attempts to introduce serum-free media resulted in prototypes with diminished stability and biological performance. The early operating assumption had been that addition of selected serum-derived proteins, growth factors and trace metals to traditional basal media would substitute for the broad cell culture functions of serum. Subsequent investigation established that, in addition to serving as a source of growth hormones, the serum additive also served various other roles that also required substitution under serum-free culture conditions to sustain normal cell growth and functionality (Jayme & Blackman 1985).

In our efforts to develop and optimize nutrient formulations for a broad range of cell types and applications, we have found it useful to classify cell culture-based applications as follows (Figure 3.1):

• cells as research and diagnostic tools to investigate normal and aberrant cell function;

• cells as biological factories to produce medicines for human or veterinary therapy; and

• cells as therapeutic products for ex vivo therapy or tissue engineering use.

Table 3.2 Motivators to reduce or eliminate serum supplementation.

Product availability

• Final product cost and impact on final dosage cost of future product

• Raw material cost fluctuation

• Finite global supply and increased demand

Downstream processing impact

• Decreased product yield and recovery

• Co-purification of serum elements with molecule of interest

Serum-associated artifacts

• Inhibition of proliferation of certain cell types by serum factors

• Induction of differentiation or apoptosis

• Proteolytic degradation of product

Regulatory concerns

• Foreign protein immunogenicity

• Adventitious agent contamination

Bioreactor Design •Stirred tank •Hollow-fibre •Microcarrier •Roller bottle •Plate bioreactor •Airlift fermenter

Culture Conditions •Cell type •Cell density •Cell cycle specificity •Campaign duration

Culture Conditions •Cell type •Cell density •Cell cycle specificity •Campaign duration

Nutrient Feeding •Batch •Fed Batch •Perfusion

Product Application •Diagnostic •Therapeutic •Regulatory environment

Nutrient Feeding •Batch •Fed Batch •Perfusion

Product Application •Diagnostic •Therapeutic •Regulatory environment

Delivery Format •Bulk liquid media •Liquid concentrates •Milled powders •Agglomerated powders •Supplements

Figure 3.1 Integrated nutrient medium optimization. Effective nutrient medium optimization for biophar-maceutical production applications cannot effectively focus exclusively on biochemical composition. Integration of formulation design optimization within process development through incorporating inputs from bioproduction, bioreactor engineering, downstream purification, regulatory affairs and business perspectives results in technical and economic superiority.

Given the title of this volume, this chapter will focus upon the second of these three categories, i.e. where cultured eukaryotic cells are used as biological factories to manufacture vaccines, interferon, and genetically engineered products (e.g. monoclonal antibodies, recombinant proteins) for human and veterinary therapy. This scope is consciously restrictive, as biomedicines have also been successfully produced in microorganisms and lower eukaryotes, as well as in transgenic animals and plants.

Recombinant proteins produced within bacterial, yeast and even insect cell production systems have typically yielded authentic peptide sequences, but also less complex post-translational modifications (e.g. glycosylation, protein folding, disulfide cross-linkages) that have rendered them less efficacious in prospective therapeutic environments. In parallel with investigation to optimize the nutrient environment for animal cell culture production applications, efforts have been made genetically to engineer lower cell types with 'mammalian-like' post-translational processing activities.

Exploitation of transgenic technologies may prove useful, particularly for production of therapeutic proteins in ton quantities. However, the potential advantages and the technical and regulatory challenges of animal and plant transgenic production systems fall outside of the scope of this book.

While this work will emphasize cell-based bioproduction applications, there also exists considerable overlap with other cell culture focal areas in terms of nutrient medium optimization requirements. Lot-to-lot consistency and absence of raw material contaminants also become critical for in vitro diagnostic applications (e.g. cytogenetics, toxicology, irritancy, carcinogenicity, immunogenicity), for high-throughput screening analysis of gene expression for drug discovery (proteomics), and for research into cellular regulatory mechanisms for expansion, differentiation and senescence.

Similarly, the ability to avoid introducing adventitious contaminants into the culture environment, or to validate their inactivation or removal during downstream purification, is relevant not just to the cell-based production of therapeutic molecules. Such assurance is also important to emerging therapies under clinical investigation where the cells themselves or living by-products are used as the therapeutic agent, such as ex vivo therapies for cancer and various immunological disorders (see Chapter 30), delivery of replacement genes or vaccines through viral vectors (see Chapters 9 and 6) and functional engraftment of genetically-engineered tissues and neo-organs (see Chapters 28 and 29).

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