Monoclonal Antibodies

Table E.1
Rankings and Priorities of Strategies for Construction of Cell Lines to produce MABs

Cell Creation Hybridoma Technology r-DNA Technology Combination of r-DNA and Hybridoma technology
Short term 1 3 2
Long term 2 3 1

Table E.2
Ranking of Technological Options for Large Scale Production of Monoclonal Antibodies

Mass production of MABs Ascites Cell Culture systems Transgenic plants and animals
Short term 1 2 3
Long term 3 1 2

Most of the research institutes contacted felt that in the short term hybridoma technology is the bet option to follow for the construction of cell lines to produce monoclonal antibodies. The next best option is the combination of r-DNA and hybridoma technology. In the case of large scale production of monoclonal antibodies, the best technology option suggested be the experts is the ascites technology followed by the controllable cell culture systems and finally by the option of using transgenic plants and animals.

In the long terms, it was felt that the combination of hybridoma r-DNA technology would be a better option to pursue for the creation of cell lines while cell culture systems would be a better strategy in the case of mass production of monoclonals. However, if volumes are going to be very large transgenic plants might be better option to follow.

A comparative evaluation of cell creation technologies and technologies for mass scale production of monoclonals is presented in Table 7.5 and 7.6

7.0 Action Plan

7.1 Requirement of monoclonals before 2000 A.D. and beyond

Scientists contacted in Indian opine that in the next 10 years, focus of research, development and commercialization should be on immunodiagnostics. They strongly recommend production of monoclonals to:

i) Diagnose infectious parasitic, bacterial and viral diseases (for example enteric fever, amoebaisis, hepatitis B virus, HIV (AIDS) virus, T.B.),
ii) Detect early pregnancy
iii) Diagnose cancer markers (ferritin, AFP, CEA) and structural defects in platelets, myloid lineage.

Besides, come scientists feel that monoclonals could be used in blood grouping, HLA typing, affinity purifications,, food processing and screening, bioactivity neutralization and as biosensors. Beyond 2000 A.D., scientist recommend using monoclonals for cancer therapy, passive immunization in parasitic, viral and bacterial infections, industrial separations, OYTC diagnostics, anti-idiotypic immunizations/vaccinations, targeted/timed release systems for drugs, vaccines, hormones, enzymes, antibodies etc and their auto-controls.

It will be desirable to develop and promote the use of semi-quantitative tests (disticks) to meet the requirements I the rural areas.

7.2 Technology Plan

Considering the above requirements of monoclonals in the two time frames (namely 1990-2000) and beyond 2000 A.D.). hybridoma technology has been considered by various experts in Indian as the technology of choice for the construction of cell lines to produce the desired monoclonals referred in section 7.1) because of

i) Its significant impacts on health care (in diagnosis of disease and monitoring of therapy) and the pharmaceutical industry and
ii) Proven technological capability in producing some monoclonals at some of the hybridoma laboratories in the country.

The following steps will have to be pursued in detail for the production monoclonal antibodies through the hybridoma technology rout:

i) Isolation of the antigenic determinant
ii) Immunization of mice which involves choice of animal, adjacent and periodicity of injections
iii) Growth of selected mouse myeloma cells
iv) Fusion
v) Screening of immunoglobulin producing hybridomas
vi) Cloning
vii) Characterization of antibodies
viii) Scale up
ix) Transfer of technology

The protocols for the important steps outlined above have been listed in Annexure 7. these protocols were obtained from the National Institute of Immunology, New Delhi, Madurai Kamaraj University, Madurai and Cancer Institute, Madras, Papers published by these institutes as well as Tata Memorial Centre, Bombay, Centre for Biotechnology Madras, Hinduja Hospital, Bombay have also been documented in Annexure 7. These papers discuss the work carried out by the various institutes for the generation of monoclonals against specific antigens. Protocols, if desired, can be obtained by directly writing to the concerned institutes.

It would also be desirable to develop the fusoma (fusion of two antibody secreting hybridomas) technology for production of bispecific antibodies. These antibodies will have wide applications in diagnosis (ELISA) and therapy. Secondly, we need to develop r-DNA technology to produce human monoclonals, and single chain/single domain antibodies for therapeutic purposes (that may be required beyond 2000 A.D.) and for developing recombinant vaccines aimed at birth control and against pathogenic bacteria and viruses (for example anti idiotypic monoclonals).

As the requirement for monoclonals increase over the years, it will be necessary to think in terms of using both the mass culture microenacapsulation technology or hollow fiber continuous flow mass culture systems and the ascitis technology. For smaller volumes and experimental purposes, the ascitis technology seems appropriate. But if the requirement goes up to 1 Kg of monoclonal antibody, then around 20,000 mice may be required. Besides problems of maintenance of large animal house facility there are related problems of contamination by virus and achievement of batch-to-batch reproducibility. Hence considering the long term requirements, it would be appropriate to import the patented ENCAPSEL or HOLOW FIBER technology and develop other related systems indigenously. On a still longer time frame, it would be important to initiate research and development projects to produce transgenic plants or genetically engineered E. Coli for large scale production of monoclonal antibodies.

It is also important to develop quality plastic ware. No locally manufactured plastic ware provides consistent and reliable binding properties, consequently these items are still being imported. R&D is necessary to develop appropriate plastic ware. CSIR labs should concentrate on finding the grade of polystyrene, the necessary additives, the moulding conditions (temperature, speed of injection, rate of cooling etc.) needed to make tissue culture grade plastic ware.

There are a number of other problems associated with the development and use of MAb kits such as cold chains, packing transportation and storage. CSIR labs should solve these problems.                                                                         Back