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Chapter 12;a Cell Culture for Commercial Settings
In the last decade, cell culture has come into its own as an area of considerable commercial importance in many biotechnology and pharmaceutical companies (Arathoon and Birch, 1986). The previous chapters have focused on techniques needed in the research laboratory, whether it is located in an academic or commercial setting. These techniques all apply to the cell culture technology that is part of the discovery of new products in biotechnology, whether it is expression cloning of a protein, purification of a desired activity using an in vitro bioassay, the production of a transgenic mouse using embryonic stem (ES) cells, or the optimization of new vectors for mammalian cell expression of recombinant proteins. This chapter will detail some special considerations that apply to cell culture as performed in commercial settings. As with previous chapters on specialized tissue culture techniques, we will not attempt to give complete details on techniques used in this setting, but will refer to published material describing techniques in each subdiscipline.
Generally speaking, the constraints on commercial tissue culture fall into three categories: (1) Speed is of essence; (2) processes must be amenable to scale up either to a rapid, automated, high-throughput format for bioassays or to large-volume production for recombinant proteins; and (3) the process and culture methods used must meet the standards of documentation, control, and reproducibility required for regulatory approval. The most efficient and successful development scientists keep these goals in mind from the beginning of the task of developing these processes and the cell lines used in them.
In this chapter we will attempt to introduce the student or scientist to a general description of how the aims of commercial cell culture can differ from common practice in a research or teaching laboratory setting. Because of commercially held trade secrets and the highly individualized nature of these processes, a complete mastery of commercial cell culture can only be gained through actual experience in a commercial setting. It should be emphasized, however, that the basic principles that control cell replication and function are the same at any scale and in any setting. The problems of adapting cell culture to commercial settings therefore is one of understanding the difference in goals (e.g., maximum titer rather than modeling of in vivo responses) and the different environments in which the cells are
expected to function. The scientific principles the industrial cell biologists use to accomplish these goals are the same as those outlined throughout this book, although the equipment used may vary.
The Cell As Industrial Property
The cells expressing a commercial recombinant protein product become a valuable asset. Newly developed or genetically modified cell lines, as well as newly developed media, may be patentable to protect the investment made in developing such tools. In addition, regulatory agencies approve the use of a specific cell line at specified passage numbers for production of recombinant or natural protein products. Thus, the description, characterization, frozen storage, and thaw of the production cell line is critical to the continued success of the product from initial development through the life of the marketed product.
Engineering Cells for Specific Properties
One great advantage of cells as living factories is the ability to select or engineer cells to express specific characteristics. This is possible even without a complete understanding of the biological pathways involved in the alterations. One familiar example is adapting cells to suspension growth. We still do not understand completely the role of substrate in regulating cell function, although there is a growing awareness of its importance and the complexity of the processes involved. Nevertheless, it is possible to select cells that will lose their requirement for attachment and grow in suspension, using procedures such as those outlined in Chapter 11. Similarly, one can adapt cells to grow in lower serum concentrations, to higher densities, and so forth. Thus, the cell may be selected to have the characteristics most desirable for use in a commercial setting. The degree to which this alters the cell's properties from those of its in vivo counterpart are important only to the extent, if any, that these alterations affect the final product to be produced. Thus, one can use the adaptability of living cells to their environment to save much time and effort in optimizing a cell culture process.
Additionally, molecular biology techniques can be used to engineer cells that have the properties desirable in a production cell. One example is the transfection of cells to allow the production of required growth factors, such as insulin, which then may act in an autocrine fashion during production runs. This leads to a less expensive and more robust culture system, since the required protein is being produced continuously in the culture. An example of this approach is shown in Fig. 12.1. In this instance, insulin was known to be required for optimal growth and secretion of protein by CHO cells. Insulin is normally made and processed from proinsulin by pancreatic beta cells. Very few cells, including CHO, can correctly process proinsulin. Therefore, in order to provide an effective autocrine growth environment, the proinsulin either must be processed or must be active without processing. The latter is the case for CHO. Therefore, cells transfected with and selected for stable expression of the proinsulin gene were able to grow as well in the absence of exogenous insulin as the parent line grew with the addition of optimal insulin (Fig. 12.1) (Mather and Ullrich, 1988). Thus, cells can be intentionally genetically altered to improve their characteristics as production cell lines. However, when using this approach, as with selection methods, care must be taken not to sacrifice optimal growth characteristics or the quality of the product when optimizing the cells for expression of a specific gene.