Antibody recombination

Herceptin HER2 receptor monoclonal antibody, recombinant... 

CD20 monoclonal antibody, recombinant [SY] MabThera [TR used by Roche outside of US and Japan]... 

While animal studies are used for safety assessment prior to administration to humans, preclinical testing may not always predict human effect. For example, continuous-infusion therapy with 260F9 monoclonal antibody-recombinant ricin A chain resulted in severe neurotoxic effects in humans that were not demonstrated in monkey toxicology studies [21], In such cases where suitable animal models are not available for safety testing it is important to consider the application of an appropriate safety factor to provide a margin of safety for protection of humans receiving the initial clinical dose [11,18]. 

In addition to ATryn , GTC Biopharmaceuticals has several other products under development in collaboration with Cento-cor, Abbott, Elan, Bristol-Myers Squibbs, Alexion, Progenies Pharmaceuticals, Im-munoGen, and Merrimak Pharmaceuticals. These collaborations and internal programs involve the production of several monoclonal antibodies, recombinant human seram albumin, a malaria vaccine, fusion proteins, and a protein for the treatment of autoimmune conditions. Some of the products that are currently undergoing cHnical trials and will be discussed later [361]. 

The main focus on modem biopharmaceuticals should be directed towards novel products which will be either monoclonal antibodies, recombinant proteins, cell and gene therapeutics, or viral products. Apart from product classes, new approaches to manufacture are also advancing, with transgenic animals and plants complementing existing cell culture-based expression (see Part IV, Chapters 5-11). (For a recent review, see Ref [8].)... 

By similar logic, protein affinity Hbraries have been constmcted to identify protein—protein combining sites, as in antibody—antigen interaction (19) and recombinant Hbraries have been made which produce a repertoire of antibodies in E. coli (20). In another case, a potential DNA-based therapeutic strategy has been studied (21). DNAs from a partially randomized Hbrary were selected to bind thrombin in vitro. Oligonucleotides, termed aptamers that bound thrombin shared a conserved sequence 14—17 nucleotides long. 

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. 

Commercial use of cell and tissue culture continues to expand. Improvement of organisms through recombinant nucleic acid techniques has become commonplace. Formerly, a few laboratories were well ahead of most others, but now the methods have been perfected for routine use. Another technique that is widely practiced is culturing of cells that excrete high concentrations of just one antibody protein. The specificity of antibodies and antigens is exploited in medical testing procedures using these pure monoclonal antibodies. 

Another application shows the preparative purification and polishing of a therapeutic fusion protein with a humanized recombinant IgG protein. The fusion protein was expressed by the fermentation of baby hamster kidney cells. The filtered culture supernatant (155 liters) contained 2.2 g of IgG and 75.5 g of total protein. After the immunoglobulins were isolated by expanded bed adsorption and rebuffering, the IgG fraction was bound to Fractogel EMD SOj (M). This column achieved baseline separation of complete antibodies (fusion protein) from small amounts of antibodies lacking the fusion part. The resulting highly purified IgG fraction (110 ml) was diluted to 150 ml and... 

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