4.0 Technological Options for India
Production of monoclonal antibodies encompasses two main phases. In the first phase, the construction of cell line or hybridoma is undertaken. This is followed by the culturing of the cells on a mass scale to produce or secrete monoclonal antibodies.
4.1 Three distinct technologies have emerged for the construction of a cell line to produce mAbs. They are Hybridoma technology, EBV hybridoma technology, combination of hybridoma and r-DNA technology.
4.1.1 The Hybridoma technology involves the fusion of antibody producing cells (isolated from the spleen of mice or rate immunized with the antigen of interest) and myeloma cells (which lacks the enzyme hypoxanthine phosphoribosyl-transferase) in the present of polyethylene glycol. Cell fusion technology which basically combines the desirable characteristics of different types of cell into one cell, is being used to incorporate in one cell the traits for immortality and rapid proliferation from certain cancer cells and the ability to produce useful antibodies from specialized cells of the immune system. Subsequent selection in a HAT medium and further cloning and recloning by limiting dilution will result in a stable cell line (hybridoma) that produces the desired monoclonal antibody. Later developments in the technology include electrofusion and laser fusion. The capital investment (only laboratory equipments) required for setting up a hybridoma laboratory in India is in the range of 15-20 lakhs of rupees. Recurring expenses for producing a monoclonal would be around 1-2 lakhs of rupees. The technical problems associated with the hybridoma technology are (i) low frequency of fusion, this can be overcome by electroporation or by using better myeloma lines (ii) weak antigens/high screening load this problem can be tackled by selection of animal species for immunization, enrichment of target lymphocytes before fusion or use of FACS for early cloning.
4.1.2 The EBV hybridoma technology involves enrichment of cells with receptors for the given antigen, immortalization of these cells by Epstein-Barr virus infection and freezing of hybrid cells for future use. Using this technology, human monoclonals have been produced to a variety of antigens including tetanus toxoid, lung tumor antigens and mycobacteria antigens. This technology has not yet been attempted in India.
4.1.3 The r-DNA technology involves the use of a novel bacteriophage lambda vector system to express in E.Coli a combinational library (light and heavy chain) of Fab fragments of the mouse antibodies. The technology developed by scientist at the research institutes of Scripps clinic, California and at the Pennyslvania State University has the potential to supersede hybridoma technology especially in producing mAbs in large amount for diagnostics, immuno purifications of therapeutic (and other biologicals) and in tailoring animal mAbs for therapeutic proposes.
Basically, the technology involves (i) harvesting of m-RNA from the spleen cells of immunized mice (ii) amplifying it using polymerase chain reaction (PCR), (ii) producing libraries of VH and VL fragments in bacteriophage lambda vector system by cloning the products of PCR amplification and by incorporating specific restriction endonuclease sites at both ends of each vector, and (v) constructing a combinational library by crossing the heavy and light chain libraries at the Eco RI site of the vector and finally expressing the desired recombinant into E. coli.
4.1.4 Combination of hybridoma and r-DNA technology involves using r-DNA technology to reproduce in bacteria to circumvent some of the problems (example hybridoma stability) associated with mAb production in mice or tissue culture. These mAbs would be free of contaminants such as viruses found in animal cells and possibility could be produced more economically in bacterial cultures than in large scale mouse ascites or cell culture protocols.
4.2 For mass scale production of monoclonals, two basic approaches are adopted. One is the invivo-such as the asciter tumors in mice or rats and the other is the invitro cell culture technology.
4.2.1 The ascites technology is adequate for small quantities of antibodies but becomes less appropriate with increase in scale. Hybridoma cells are injected into the peritoneal cavity of histocompatible mice (which have been primed with pristone) and the ascites fluid which is formed after 7-10 days is collected in heparin. The ascites fluid is centrifuged to remove lipids and small fibrin clots. Antibodies are then precipitated out by the addition of equal volume of ammonium sulphtae. The dialysate contains about 80% of antibody which may be further purified or used directly. Several companies in the U.S. own well established mice breeding colonies and provide ascites fluid containing 2-10 mg/ml of mAb at a cost of $3-4 per ml or <$1000 per gm) of mAb. In India, Ranbaxy Laboratories, Delhi and Lupin Laboratories, Bhopal have set up animal houses to use the ascites technology to produce mAbs.
4.2.2 The in vitro cell culture technologies can be broadly distinguished into technologies in which the cells are immobilized or entrapped and those in which the cells are in free homogenous suspension. In the airlift technology (suspension culture technology), gas mixtures are introduced from a sparge ring into the base of a concentric draught tube within the airlift vessel. The gas hold up in the draught tube causes a difference in density between the contents of the draught tube and the outer zone of the vessel which establishes circulation of the culture. The pH and dissolved O2 are controlled automatically in the vessel. The hybridoma cells secrete antibody into the surrounding medium during the growth, stationary and decline phases of the growth cycle. Antibody production varies in the range of 40-500mg per liter with an average of 100 mg per litre for all cell lines. The clarified supernatant is then rapidly concentrated by tangential flow ultra filtration to give a concentrate training 1 gm of antibody per liter. This is slightly less than the concentration obtained in the ascites technology. Serum free media has now been developed for use in air lift fermentor which give yield comparable wit those seen in serum supplemented media. Normally yields of 2gm/litre of antibody have been achieved.
4.2.3 Microencapsulation technology has been demonstrated as an efficient system for large scale production of both human and murine monoclonals of high purity and activity. Micro encapsulation technology uses a porous carbohydrate capsule to surround the hybridoma cells and to retain the antibodies while allowing the circulation of nutrients and metabolic wastes. After 2-3 weeks in culture, the encapsulated colonies are harvested and washed to remove the growth medium. When the capsule is opened, mAbs concentration is found to be in the ranger of 45-80%. Cell densities of greater than 5 x 108 cells/ml are obtained. Final concentration of antibody range from 0.5 to 3 mg/ml depending on the cell line and its rate of antibody production. Approximately, 5-20 gms of antibody can be produced by one 40 L fermentor. Antibodies with 98% purity can be obtained by ion exchange chromatography. Antibodies produced by Micro encapsulation are being utilized in diagnostic assays, in large scale purification of biologicals and for invivo imaging and the treatment of human cancer and other diseases. This technology has been patented by Damon Biotech in 1982. The yield obtained is in the range of 1-10g per liter.
4.2.4 Hollow fiber perfusion reactor technology is well suited for long term continuous culture. It has many advantages for large scale mammalian cell culture – high cell densities (>108 cells/ml), efficient distribution of nutrients and removal of metabolic waste products, immobilization of cells in the fiber bundle allowing cell-free harvests. Hollow fiber reactors have been used since the early seventies. The hollow fiber (microprorous membrane) unit contains viable cells maintained in the extra capillary space surrounding the fibres. Most reactors use medium flowing through the fiber humers to support cells in the spaces between the fibres. The hollow fiber capillaries pass through the centre of the chamber and deliver a constant influx of freshly oxygenated medium with low level defined protein components selected to optimize production of antibody. Yields of 2 g per liter have been obtained and a retail cost of $1000 per gm of antibody is claimed. The perfusion technology will provide a viable means to address the expected markets from mAbs because of its low protein defined media, high cell densities and longer production runs for effective scale up.
4.2.5 The ceramic matrix technology is based on the immobilization of the hybridoma culture on a ceramic matrix which is encapsulated in a set of plastic end cps that allows medium to floe through the matrix. The total cell immobilized in the system range between 3 and 10 x 1010 cells depending on the cell line and day of harvest. Opticell has obtained a yield of 0.3 g per litre at an estimated cost of $ 1200-$2500 per gm. of antibody.
4.2.6 Production of mAbs by transgenic plants will become significant for large scale production of mabs by 1995. genetically stale seed stocks of antibody producing plants can be isolate and stored indefinitely at low cost and the seed stock can be converted into a harvest of any quantity of antibody within one growing season.
The cost of agriculturally produced antibodies is likely to be considerably less than antibodies produced from hybridoma cell cultures or ascites fluid. Researchers at Scripps Clinic, U.S.A. claim that if antibodies were expressed in soybean and constituted 1% of total protein in soybean meal, a kilogram of antibody could hypothetically be produced for less than $100. Agricultural production of mAbs will be flexible, high capacity and cheap. This will encourage isolation of therapeutic antibodies.
4.3 Purification of mAbs by affinity chromatography offers excellent purification in a single step even if the sample is very dilute. It involves separation based on irreversible immobilization on a solid phase matrix (usually agarose). The method is rapid and requires minimum investment. Almost 100% purity is obtained with yield typically between 50 and 80%. Ion exchange chromatography on DEAE cellulose is an expensive and effective method for purification of lgG lgM. This method has good scale-up potential, but the degree of purity attainable at a large scale is rarely above 70% for a single step process.