Mammalian cells with antibodies

The development and commercial success of the first monoclonal antibody used to treat tumors in the United States have had broad consequences. Companies have pushed ahead with the development of a variety of additional monoclonal antibodies. Six years after rituximab was approved, more than one third of the 38 approved recombinant proteins manufactured in mammalian cells were antibodies (some of the most notable are listed in Table 32.1). Many of these antibodies are used at large doses that were not thought to be economically practical 10 years ago. Today, the pharmaceutical industry is facing a projected shortage of manufacturing plants for recombinant proteins synthesized by mammalian cells, and there are still more than 400 monoclonal antibodies reported to be in clinical trials. 

Ricin, a type II RIP found in the bean of the castor plant, has been extensively used for targeted tumor therapies replacing the lectin B chain, which binds to almost all mammalian cells, with peptides, antibodies, or proteins targeting cancer cells leads to selective target toxicity [105]. Several clinical trials by this approach have been conducted (see Section 7). 

Since its first appearance, the technique has successfully been used in many different applications (Potter, 1988, 1993 Neumann et al., 1989 Chang et al., 1992 Shikegawa and Dower, 1988 Chassy, 1988) transient and stable transfection of bacteria, plant, and mammalian cells with exogenous DNA (electrotransfection) insertion of enzymes, antibodies, biochemical reagents, viruses, and particles into cells deposition of macromolecules in cell membranes and electrofusion of whole cells. 

Product formation kinetics in mammalian cells has been studied extensively for hybridomas. Most monoclonal antibodies are produced at an enhanced rate during the Gq phase of the cell cycle (8—10). A model for antibody production based on this cell cycle dependence and traditional Monod kinetics for cell growth has been proposed (11). However, it is not clear if this cell cycle dependence carries over to recombinant CHO cells. In fact it has been reported that dihydrofolate reductase, the gene for which is co-amplified with the gene for the recombinant protein in CHO cells, synthesis is associated with the S phase of the cell cycle (12). Hence it is possible that the product formation kinetics in recombinant CHO cells is different from that of hybridomas. 

Feuser, J., Halfar, M., Liitkemeyer, D., Ameskamp, N., Kula, M.-R., and Thommes, J., Interaction of mammalian cell culture broth with adsorbents in expanded bed adsorption of monoclonal antibodies, Process Biochem., 34,159,... 

One of the most obvious benefits of plants is the potential for production scale up, leading to the production of virtually limitless amounts of recombinant antibody at minimal cost Plants are easy to grow, and unlike bacteria or animal cells their cultivation is straightforward and does not require specialist media, equipment or toxic chemicals. It has been estimated that plantibodies could be produced at a yield of 10-20 kg per acre at a fraction of the cost associated with production in mammalian cells [2,18] The use of plants also avoids many of the potential safety issues associated with other expression systems, such as contaminating mammalian viruses or prions, as well as ethical considerations involving the use of animals. 

Mammalian cell suspension cultures are the preferred choice for large-scale recombinant protein production in stirred-tank bioreactors. The most widely used systems are Chinese hamster ovary (CHO) cells and the murine myeloma fines NSO and SP2/0. In half of the biological license approvals from 1996-2000, CHO cells were used for the production of monoclonal antibodies and other recombinant glycosylated proteins, including tPA (tissue plasminogen activator) and an IgGl fusion with the tumor necrosis factor (TNF) receptor, the latter marketed as Enbrel [7]. 

For other plant-derived antibodies, stability was shown to be similar to mammalian counterparts. For instance, a humanized anti-herpes simplex virus monoclonal antibody (IgGl) was expressed in soybean and showed stability in human semen and cervical mucus over 24 h similar to the antibody obtained from mammalian cell culture. In addition, the plant-derived and mammalian antibodies were tested in a standard neutralization assay with no apparent differences in their ability to neutralize HSV-2. As glycans may play a role in immune exclusion mechanisms in mucus, the diffusion of these monoclonal antibodies in human cerival mucus was tested. No differences were found in terms of the prevention of vaginal HSV-2 transmission in a mouse model, i.e. the plant-derived antibody provided efficient protection against a vaginal inoculum of HSV-2 [58]. This shows that glycosylation differences do not necessarily affect efficacy. 

Carboxypeptidase G2 (CPG2, glutamate carboxypeptidase, EC 3.4.17.11) is a bacterial enzyme not produced in mammalian cells. It has been used with promising success to target cytotoxic alkylating agents to tumor cells [60][61]. In a series of studies, a CPG2 monoclonal antibody con-... 

Host Cell Impurities Various organisms have been used to produce recombinant proteins yeast, bacteria (e.g., E. coli), insect cells, and mammalian cells such as Chinese hamster ovary (CHO) cells. During the purification process, some HCPs can copurify with the protein product. Because of the specificity of the antigen-antibody interaction, an ELISA can be used to detect and quantitate the contaminating HCPs. Detecting host impurities is important for quality process control as well as for product safety issues. The intent is to avoid unsafe levels of residual HCPs which might lead to adverse reactions.11... 

The modern biotechnology industry has come up with important blood products, infectious disease combatants, immunotherapy products, growth factors for mammalian cells, and monoclonal antibodies. Approximately 4600 biotechnology companies exist in the world. Over 140 biopharmaceuticals are on the market and hundreds more are in the clinic against over 200 diseases (AIDS, cardiovascular, diabetes, arthritis, cancer etc). 

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