Animal Cell Technology

From Biopharmaceuticals to Gene Therapy

Leda Castilho, Angela Moraes, Elisabeth Augusto, Mike Butler, Leda Castilho, Angela Moraes, Elisabeth Augusto, Mike Butler

• Publisher: Taylor & Francis
• Year: 2008
• ISBN:
  9781134099719
• Edition: 1
• Topic: Medicina
• Subtopic: Biotecnologia in medicina

Animal Cell Technology: from Biopharmaceuticals to Gene Therapy provides a comprehensive insight into biological and engineering concepts related to mammalian and insect cell technology, as well as an overview of the applications of animal cell technology. Part 1 of the book covers the Fundamentals upon which this technology is based and covers the science underpinning the technology. Part 2 covers the Applications from the production of therapeutic proteins to gene therapy. The authors of the chapters are internationally-recognized in the field of animal cell culture research and have extensive experience in the areas covered in their respective chapters.

1 Introduction to animal cell 1 technology

Paula Marques Alves, Manuel Jose´ Teixeira Carrondo, and Pedro Estilita Cruz

1.1 Landmarks in the culture of animal cells

Despite the dominance of animal cell culture in the production of biopharmaceuticals in recent times, this technology was not consolidated into standardized large-scale bioprocesses until the 1990s. Nevertheless, the first experience with animal cell culture can be traced back to the beginning of the 20th century. By the use of the hanging drop technique and frog heart lymph, Ross Harrison, at Yale, tried between 1906 and 1910 to elucidate how the nervous fiber is originated (Witkowski, 1979). He considered three hypotheses: (i) in situ formation from the nerve sheath; (ii) preformed protoplasmic bridges; or (iii) as a result of the nerve cell growth itself. When Harrison demonstrated the validity of the third hypothesis, he also confirmed the cell as the primary developing unit of multicellular organisms.

An early pioneer of cell culture was the French surgeon Alexis Carrel, who won the Nobel prize in Medicine in 1912 for his research at the Rockefeller Institute (Spier, 2000). Harrison was, above all, the inventor of analytical solutions, while Carrel, with his extensive clinical practice experience, sterility concerns, and capacity to develop appropriate culture media and culture flasks, created the change in the technological paradigm that led to the start up of animal cell technology. By careful manipulation, Carrel insured the maintenance of chicken embryo cells for several decades in culture.

Spier (2000) lists some essential differences between cells in an organism (in vivo) and cells in culture (in vitro), particularly the following;

• Tissues are three-dimensional, while cell cultures are of zero dimension (monodispersed in suspension culture) or two-dimensional (monolayer growth). However, some culture techniques exploit three-dimensional systems (Alves et al., 1996; Powers et al., 2002).
• In tissues, cells are subject to tension and compression, but not when in culture, with the exception of artificial organs.
• In tissues, lymphokines and chemokines vary in proportion and concentration to allow fluctuations of short (cardiac rhythm), medium (daily), and long duration (life cycle). However, in culture these parameters normally do not vary.
• The mechanisms for cellular differentiation control in tissues and in culture are distinct.

The need to deal with some of these differences demands enormous efforts for the development of culture media (chemical environment) or shear, mixing, viscosity and bubbling conditions (physical environment), which should be optimized to result in an industrial process that can be validated. Requirements to avoid contamination have led to the formulation of serum-free media or even of protein-free, chemically defined media for the production of biopharmaceuticals (Griffiths, 1988).

The range of culture flasks and reactor types employed is quite wide, both for suspension and adherent cultures, from small Carrel’s or Roux’s flasks to roller bottles. Fixed- and fluidized-bed bioreactors, air-lift reactors and even stirred and aerated tanks with capacities up to 15 m3 are common in large plants producing monoclonal antibodies (mAbs) for anticancer therapies (Adams and Weiner, 2005; Griffiths, 1988).

One of the main purposes of animal cell culture development was the search for viral vaccines, initiated during the Second World War (1939– 1945), particularly for poliomyelitis. The names of Enders, Syverton, and Salk are undoubtedly associated with the production of the inactivated polio vaccine, approved in the USA in 1955 and produced on a large scale in primary monkey kidney cells. Later, after a dispute between Hilary Kaprowski and Albert Sabin, the attenuated vaccine against poliomyelitis was licensed in 1962. At the end of this period, at the Wistar Institute, Hayflick developed a cell line from embryonic tissue capable of replicating more than 50-fold before becoming senescent (Hayflick and Moorhead, 1961). The cell was diploid, easy to freeze and to reactivate and did not show any evidence of contamination by the viruses normally found in monkey primary kidney cells. This cell line (WI-38) turned into the basis for the production of human viral vaccines against poliomyelitis and MMR (measles, mumps, rubella), while other cell lines were evaluated for the production of veterinary vaccines, such as BHK (baby hamster kidney) in the case of the vaccine against foot-and-mouth disease.

After this period, there was an accelerated use of animal cells. Namalwa cells (Finter et al., 1991) were used to produce alpha-interferon by Well-come in 1986. Vero cells (a cell line derived from monkey) were used for anti-rabies vaccine. Hybridomas were considered acceptable for mAb production and the use of genetically modified CHO (Chinese hamster ovary) cells was authorized for the production of tissue plasminogen activator (tPA) by Genentech in 1986.

Finally, three relevant aspects should be mentioned to clarify the scientific, technological, and industrial position of biopharmaceuticals and animal cells.

• Complex biopharmaceuticals, such as proteins, virus or virus-like particles (VLPs), among others, produced by cellular and/or recombinant technologies are characterized/defined by their own production processes. This means that analytical, biological and immunological characterization assays are usually not considered sufficient for product marketing, given the complexity of the molecules. Therefore, product licensing is based on the specific production process, which cannot be altered. Process changes may require new licensing proce- dures, and this makes the introduction of biosimilars in the market more difficult.
• Some of these proteins (for therapy) or VLPs (for vaccines such as hepatitis B) can be produced by yeast or even by Escherichia coli because of the limited requirement for post-translational modification (see Chapter 6). The total market value of biopharmaceuticals produced by E. coli or yeast was surpassed by those produced by animal cells only around 1996. In the last few years, market dominance in favor of animal cells has increased significantly.
• Considering the complexity and instability of biopharmaceuticals, the production process from animal cells has to be designed, modeled, and optimized in an integrated form, taking into account the culture, extraction, and separation (Cruz et al., 2002). This may be different from what is normal in the production of simple biological compounds such as antibiotics or vitamins.

The number of biological processes has increased tremendously in the last 15 years. This has resulted in high expectation for an improvement of quality of life and an increase in the volume of business related to products obtained from animal cell culture technology, with the broad potential use of these products for disease diagnostics, prevention, therapy, and cure. Table 1.1 indicates some examples of approved therapeutic products obtained through animal cell culture.

1.2 Types of animal cell cultures

The methods developed for obtaining and maintaining primary cultures paved the way for animal cell technology. However, the huge growth and expansion of this technology was possible only because new cell types were established, namely diploid cells, hybridomas, and other continuous cell lines. Animal cells in culture can be classified, according to their origin and biology, into primary cultures and cell lines (nomenclature most frequently employed in textbooks). However, depending on their applications, animal cells can also be grouped as follows.

Table 1.1 Examples of approved products obtained through animal cell culture

• Cells producing proteins employed in the production of complex therapeutics, subunit vaccines, and diagnostic products, such as CHO, BHK, HEK-293, WI-38, MRC-5, SP2/0, NS0, and insect cells.
• Cells producing viruses used in gene therapy and viral gene vaccines (for instance, Vero, HEK-293, and PER.C61 cells).
• Normal cells, tumor cells, and stem cells used in research and development, specifically in the discovery of new products and for in vitro study and toxicology models (e.g. nerve cells, fibroblasts, Caco- 2, MRC-5, and endothelial cells).
• Human cells for subsequent use in cell therapy and regenerative medicine (e.g. embryonic and adult stem cells).

Primary cells are isolated directly from organs or tissues. Primary cells are normally heterogeneous and better represent the tissue from which they originate. These cells have a finite growth capacity and can be subcultured for only a limited number of passages. Subcultured cells, which have been selected to form a population of cells of a single type, are designated cell lines, and can be finite or continuous. Finite cell lines (cells capable of a limited number of generations before proliferation ceases) as well as continuous cell lines can be propagated and expanded for the production of well characterized cell banks, where they are preserved by employing cryopreservation techniques (Doyle et al., 1994).

A normal tissue usually provides finite cultures, while cultures obtained from tumors can result in continuous cell lines (immortal). Nevertheless, there are many examples of continuous cell lines that are obtained from normal tissues and are not tumorigenic, such as BHK 21 (baby hamster kidney fibroblasts), MDCK (Madin-Darby canine kidney epithelial cells), and 3T3 fibroblasts (Freshney, 1994, 2000). Immortal cell lines can occur spontaneously (rarely) or after a transformation process (more often), which can be induced by carcinogenic chemical agents, by viral infection, or by the introduction in the cell genome of a viral gene or an oncogene capable of overcoming senescence. Several of the differences between normal and neoplastic or tumor cells are analogous to the differences between finite and continuous cell lines, since immortalization is an important component of the cell transformation process.

The main advantages of continuous cell lines are: (i) faster cell growth, achieving high cell densities in culture, particularly in bioreactors; (ii) the possible use of defined culture media available in the market, mainly serum-free and protein-free media; and (iii) the potential to be cultured in suspension, in large-scale bioreactors.

The major disadvantages of these cultures are the accentuated chromosomal instability, the larger phenotype variation in relation to the donor tissue, and the disappearance of specific and characteristic tissue markers (Freshney, 1994).

Many examples of immortalization methodologies and techniques to obtain continuous cell lines are described in the literature (Land et al., 1983; MacDonald, 1990; Ruley, 1983), including those involving transfection or infection with viral genes (for instance, the E6 and E7 genes of human papilloma virus, and the SV40T simian virus 40 large T-antigen gene) or virus (such as Epstein–Barr virus and retroviruses). Another strategy is to create hybrid cells resulting from the fusion of a cell with a limited lifespan with a continuous cell. This is the strategy used to obtain hydridomas for antibody production, as discussed below (see also Chapter 17).

The hybridomas were, to a large extent, responsible for the biotechnology ‘‘explosion’’ towards the end of the 1970s, opening perspectives for remarkable advances in both the immunotherapy and diagnostic areas. In 1975, Ko ¨ hler and Milstein demonstrated that, despite the impossibility of cultivating differentiated B lymphocytes in vitro, it was possible after their fusion with immortal myeloma cells. The hybrid cell lines (hybridomas) can grow continuously and produce and secrete immunoglobulins. Since all the immunoglobulin produced derives from a single type of cell, the antibody is monoclonal and is directed against only one epitope. Although the initial studies performed by Ko ¨ hler and Milstein were restricted to the production of mouse mAbs, soon thereafter the production of antibodies from other species, including human, became possible.

As mentioned before, another relevant key development for the progress of animal cell culture technology was the WI-38 human diploid cell line obtained by Hayflick and Moorhead in 1961, since previously the options were the use of primary cultures or of heteroploid cell lines (derived from tumors or from cells that acquired tumor-like characteristics in culture). Because of that, heteroploid cells were not acceptable for the production of compounds for human applications, and therefore primary cultures from other species were employed (such as primary monkey kidney cells). After the development of diploid cell lines, i.e. with a diploid karyotype, a new concept of cell line emerged, since these cells are considered ‘normal’ cells. They undergo senescence and die in culture after a finite number of generations (around 50 generations for WI-38 cells). The disadvantages of these cells in culture are that they grow slowly, not reaching high cell densities, present relatively low productivity, are highly dependent on support adhesion for growth, and consequently are not easily cultured in suspension. Nowadays, the diploid cell line most frequently employed is MRC-5, and its use is particularly important for the study of cell aging mechanisms.

The animal cell lines mentioned above are more extensively discussed in Chapters 2, 17, 18, 20, and 21.

1.3 Use of animal cells in commercial production

1.3.1. Animal cell proteins in human diagnosis and therapy

mAbs are currently the most important class of pharmaceutical proteins in terms of market volume. Given their enormous biological specificity it is not surprising that their first clinical applications were the so-called immunoassays, such as the ELISA-type assays, for in vitro diagnosis. After 1980, however, mAbs also started to be used in association with radio- active markers, in imaging methods, such as immunoscintillography. In addition, with higher doses of the radioactive agent used for tumor detection, it became possible to treat cancer. This is the ‘magic bullet’ concept initially proposed by Paul Ehrlich at the end of the 19th century. In this application, the antibody directs the radioactive product only to cancer cells, which express large amounts of surface tumor antigens. Instead of radioactive compounds, lymphokines or toxins can also be associated to the antibodies to cause the death of tumor cells. The first therapeutic antibody approved (Orthoclone1 OKT-31 or

Muromonab CD3, 1986) was indicated not for cancer treatment, but for controlling acute rejection of transplanted organs (kidney, heart, and liver). Nowadays, other clinical indications such as asthma, rheumatoid arthritis, psoriasis, and Crohn’s disease are treated with mAbs (see Chapter 17) (Antibody Engineering and Manufacture, 2005; Monoclonal Antibodies and Therapies, 2004; Hot Drugs, 2004; Walsh, 2004).

Many recombinant proteins that are not antibodies are also on the market for distinct applications (see Chapter 16). Examples are factor VIII for hemophilia A treatment (Bayer, 1993, produced from BHK cells), erythropoietin as an anti-anemic agent (Amgen, 1989, produced from CHO cells) and μ-interferon for the treatment of multiple sclerosis (Biogen and Serono, 1996, produced from CHO cells).

Nevertheless, in the last 20 years, the relevance of clinical diagnosis has increased significantly, forming the basis for the pharmaceutical strategy known as pharmacogenomics, which could in the future enable a complete customization/individualization of pharmaceuticals. A direct connection between diagnosis and treatment could make it possible to introduce individualized products that were eliminated for general use due to side effects that were severe, but occurred only in a limited number of patients. This would be acceptable provided that unequivocal diagnostics can be developed to identify these patients. This process has already been adopted sometime ago, for instance, to guarantee that patients who are allergic to an antibiotic, such as penicillin, are not treated with it. However, individualized medical treatment may become more and more frequent to insure that expensive biopharmaceuticals are administered only to patients who can benefit from them and avoiding excessive health expenses. One example is the biopharmaceutical ‘trastuzumab’ (Herceptin1, Genentech),example is the biopharmaceutical ‘trastuzumab’ (Herceptin1, Genentech), used for the treatment of a very aggressive breast tumor type that over-expresses the epidermal growth factor receptor type 2 (HE...