Monoclonal Antibodies

Code No: TMS029 Price: 1100 Category: Biotechnology

 

Summary  : The relevance of monoclonal antibodies to the broader area of biotechnology and other sectors is surveyed and the emerging markets and technologies (Worldwide) and in India is assessed.
The technological options available to India in the social and techno-economic context on the basis of impact analysis.
The study finally presents an action plan to implement the preferred technology options.

 

Table Of Contents :

  • Objective of the study
  • Conceptual and methodological framework
  • Biotechnology
  • An overview - Antibodies and overview
  • Monoclonal antibodies; properties and application
  • Production and markets for monoclonal antibodies
  • Worldwide and India
  • Technological options
  • Evaluation of technological options
  • Action plan.


1.1 Relevance of Biotechnology to Economy, Industry and Society

Developments in biotechnology have wide ranging impacts on the economy, industry and society at large. The fastest growing biotechnology market is that for diagnostics (monoclonal antibodies, biosensors, gene probes) which can be applied to humans, animals, plants the environment and industry. New diagnostic tests will lead to a revolutionary change in medical practice. The rapidity, specificity and facility of use of new diagnostic kits ushers in an era of mass diagnosis extending to the general population and reducing the damage caused by disease leading to an improvement in quality of life, with human and social benefits much larger than the direct economic benefits.

Biotechnology can accelerate the development of low cost inputs in livestock production and animal health coverage e.g. early diagnosis, prevention and prophylactic measures for overall improvement in livestock health in rural areas which are a crucial link in economic development.

The new trends will also have impacts in the pharmaceutical industry which will change from a supplier of products to a health care industry (i.,e. a supplier of a wide range of diagnostic and therapeutic products, auxiliary materials, and biomedical systems). The massive diffusion of diagnostics will move the current process of transformation towards an extended multidisciplinary base and an integration of biotechnology, microelectronics and telecommunication.

1.2 Present Status of Technologies in the World and in the India in the area of Biotechnology

Major technological changes in the world are in (i) the development of r-DNA technology based on the powers of gene cloning and splicing which allow for the production of large quantities of DNA and (ii) utilization of hybridoma technology which allows for the fusion of specific antibody producing spleen cells with myeloma cells to produce large quantities of pure antibodies. In India, the hybridoma technology is being used in several research laboratories attached to hospitals and national institutes for the production of diagnostic kits. The r-DNA technology is being developed in few of the research organizations for the expression of DNA against specific proteins. In the field of agriculture, cell fusion and tissue culture technologies are being used to target gene products into plants to obtain high yield, pest resistant hybrid seeds and plants.

1.3 Technological gaps in Biotechnology Research

The technological gaps that need to be addressed are in the area of developments of r-DNA technology, integration of disciplines like genetics, immunology, bioprocess engineering, information technology and other basic areas of science like physics, chemistry and biology.

1.4 Present Status and Projection of Markets – World wide and India in the Area of Biotechnology

The mainstay of biotech activity for the next 10 years will be human health care – diagnostic and therapeutic products. According to some global market estimates, monoclonal antibody products (mainly diagnostics, therapeutics and industrial separation agents) are expected to grow from about $1 billion sales in 1989 to about $ 8 billion by year 2000 A.D. This would comprise about 15% of the total biotechnological products available in the world market.

According to available data on projections of biotech markets in India, the market for biotech products is expected to grow to Rs. 3500 crores in India by 2000 A.D.                                                                                                                 Back


2.0 What are Antibodies

2.1 Antibodies, protein molecules produced by specialized cells (B Lymphocytes, spleen, lymph glands and blood) in the body, are a basic constituent of animal and human diseases fighting immune systems, that bind to antigens thus effectively neutralizing and destroying them. Antibodies are symmetrical molecules made up of four polypeptide chains containing two identical glycosylated heavy chains and two identical non-glycosylated light chains. The basic unit structure is folded in such a manner to form on its surface two identical regions consisting of heavy and light chain called Fab (antigen binding sites) and a third region called Fc which determines the biological properties. Based on physiochemical and antigenic differences. Five classes of antibodies or immunoglobulins have been recognized: lgG, lgA, lgM, lgD, and lgE. lgG is the major immunoglobulin component of serum making up 75% of the total. When molecules of antibody and antigen are brought together in solution, they interact with each other by forming a link between the antigen binging site on the Fab fragment of the immunoglobulin and the particular chemical groupings which make up the antigenic determining site on the antigen molecule.

2.2 Antigen- Antibody Reactions

Antigen–antibody reactions play an important role in the diagnosis of disease and the in the identification of microorganisms. The key antigen-antibody reactions are agglutination, precipitation, complement fixation. Radio immunoassays and immunoassays using enzyme–linked antibody or antigen are presently used in diagnostic test.

2.3 Monoclonal Antibodies, Polyclonal Antibodies and Single Domain Antibodies

Monoclonal Antibodies (mAbs) are antibodies secreted by a clone of cells. The most popular method of obtaining a clone of such secretors is b fusing a myeloma cell (an immortal B cell preferably a non-secretor) with B cells from the spleen. The resultant hybrids are cloned and each clone produces antibody all of which have the same binding specificity and affinity.

Because they are more specific and sensitive than other antibodies, they can used in diagnostics kits to detect the presence of viruses, bacteria, parasites, chemicals ad biologicals.

Monoclonal antibodies are the reagents of choice when

1) The antigen is difficult to purify (as in the case of lymphocyte markers)
2) The antigen is proteinaceous in nature and
3) There are risks involved in handling the serum (as is the case of anti-blood group sera obtained from professional donors)

The technology to produce mAbs was developed in 1975 by Cesar Milsterin. This was achieved by fusion of myeloma cells with lymphocytes from spleen of mice immunized with particular antigen. The hybridoma so developed has the ability to multiply indefinitely and to produce an antibody of predetermined specificity.

Polyclonal Anti bodies (pAbs) are a mixture of many antibodies with varying affinities and specificities. The cost of development of pAb may be small but its requirement fro purified antigen for its production increases its actual production cost.                                                                                                                                                                Back


 

Application areas suitable for use of polyclonal antibodies are as follows:

a) Sandwich ELISA for tumor markers or other antigens can be designed with polyclonal antibodies as the coating (trapping) antibody, followed by addition of standard antigen/sample and then addition of relevant MAb conjugated to HRPO.
b) Polyclonal antibodies are useful in histopathological analysis using immunoperoxides staining technique.
c) In some cases of affinity purification of antigens, polyclonals have advantages over MAbs.

Single domain antibodies (dAbs) was developed by scientists at LMB, Cambridge. They consist of just the binding site of the orginal antibody and are produced by injecting the relevant genes, take directly from the mouse, into the bacteria. The small size of dAbs gives them the ability of penetrate tissue quickly, block specific active sites on a virus and rapidly diffuse. Their cheap production (100-10000 times less than mAbs/pAbs) is a major advbantage3. According to Scotgen, U.K., dAbs will not appear in products until the latter half of the decade and will displace mAbs in some applications by year 2000 A.D.

MAbs are used instead of pAbs because of better specificity, sensitivity, ease of applicability and use and availability of instrumentation for automation.

2.4 Bispecific, Chimaeric and Single Chain Antibodies

Bispecific Antibodies are antibodies in which the combining sites of the immunoglobulin molecule react with two distinct antigenic determinants. This property is very useful in therapy and diagnosis. In in vitro diagnosis, bispecific antibodies provide higher signal-to-noise ratios, and the possibility of multiple antigen detection. In therapy, they are less toxic, more active and have a longer half-life.

Chimaeric Antibodies can be obtained by coupling human CH (constant heavy) and CL genes (constant light) with mouse VH (variable heavy) and VL (variable light) genes and then introducing them into myeloma cells. Such combinations may be more therapeutically useful than mAbs of purely murine origin, because of less antigen city. Chimaeric antibodies have potential in the treatment of cancer, auto immunity, graft rejection and infectious diseases where repeated administration of antibody reagents may be required.

Single Chain Antibodies are produced by genetically linking heavy and light chain variable region genes with a DNA sequence encoding a synthetic peptide linker. These antibodies offer reduced toxicity, improved attachment of imaging or therapeutic agents and improved binding performance when immobilized. Production of single chain antibodies are expected to be low cost because the genetically engineered organism can be grown rapidly in large volume fermentors on in expensive media. No products of this technology are expected to reach clinical research by 1992.

3.1 Importance and relationship of monoclonal antibodies to the broader area of biotechnology and other relevant sectors

Monoclonal antibodies is a significant branch of biotechnology that has a pervasive impact on the health care industry and is likely to play a major role in diagnostics, therapeutics and industrial purification of biological and chemical products. Early diagnosis of disease will have a significant impact on (i) quality of life for the patient (ii) survival rate (iii) health care costs. Detection of crop diseases can aid the farmer to use a more specific type of herbicide or fungicide in a smaller dose. This will not only increase the yield but also reduce the cost of raising crops.

3.2 Status of ongoing research in India for development and production of mAbs

National Institute of Immunology (NII), Delhi has reported generation of mAbs to detect typhoid fever, and hepatitis B virus. They are in the process of developing monoclonals against rotavirus and virulent stains of E. Coli. This is under clinical trials. Monoclonals to detect bacteria that are difficult to culture or have very long incubation periods in vitro such as Mycobacterium spp. Brucella spp. Are being developed but research has not been successful so far. Research work in India is yet to be initiated for the diagnosis of life threatening infections such as streptococcus group B in the central nervous system, vibrio cholera infection.

Monoclonals have been raised against variety of antigens related to human medicine including hormones, proteins involved with a tumour, drugs. A pregnancy test kit which detects human chorionic gonadotropin (HCG) in the urine of pregnant women has been developed in India by Ranbaxy laboratories in collaboration with NII, Delhi. These kits are already available in the market. Lupin Laboratories has set up a project 5 years ago in Bhopal to produce monoclonal diagnostic kits using indigenous hybridoma technology and ascitis technology. Some of the kits are undergoing clinical trials. In the second phase they plan to use r-DNA technology to produce therapeutic drugs, hepatitis vaccines and growth hormones. Span Diagnostics, Surat drugs, hepatitis experimenting with the preparation of ELISA kits for Hepatitis B antigen. Monoclonals have also been generated against cancer markers CEA, AFP, feritin by cancer institutes in Madras and Bombay. Indian Institute of Microbial Technology (IMTECH) Chandigarh has developed monoclonals against urokimase, fibrin, rifamycine oxidase, tissue type plasminogen activator. Tata Institute of Fundamental Research (TIFR), Bombay has developed mAbs against neural antigens of Drosophila and Zebra fish and nitrate reductase. P.G. Institute for Medical Research, Chandigarh has developed antibodies against human sperm antigen and leishmania donovani.                                                                                                     Back


 

3.3 Applications and markets for Monoclonals Abroad

Detection of tumors in vivo has been attempted by the use of radiolabel led antibodies. Immunoassays are available abroad for micro biological and chemical analysis of food, for example the salmonella screen kit, test kit for toxin contaminant of food. MAbs are increasingly being used in immuno affinity chromatography (IAC) of therapeutic proteins, although use of mAb based IAC in the preparation of pharmaceuticals for parenteral use is till in its infancy. During the last 2 years, mAbs with enzyme like catalytic properties have been successfully raised and their applications will be seen in biosensors which can be used for monitoring of body functions during intensive care, effluent monitoring and in process control in pharmaceutical and chemical industries.

Monoclonals have been produced against dioxin and tetanus toxin. In additional to potential clinical utility, monoclonals to tetanus toxin have been used in vitro to aid in the localization of cells and tissues reacting to tetanus toxin. Monoclonals have also been produced for drug targeting.

According to Business Communication Co. U.S., Polyclonal antibodies accounted for 71% of the antibody products available world wide in 1988 while monoclonals accounted for abut 29% of the antibody products in the same year when the world sales of antibody products amounted to some $1082 million. Diagnostics utilizing monoclonal antibodies and DNA probes have been the fastest growing components of new biotechnology, accounting for more than half of the current market for new biotech drugs. It is predicted that by 1992, mAbs will have replaced virtually all diagnostics now based on polyclonal antibodies. At least 220 diagnostic kits using mAbs and 8 using DNA probes were available in 1989. AIDS testing alone was valued at $75 to $100 million in 1987 and is growing rapidly. The value of output of this emerging industry now represents around 1.5% of the pharmaceutical industries present commerciali-zed output. Analytical applications account for 85% and separation applications account for 10% of the total sales of antibody products. It is expected that the total monoclonal market might reach $8 billion by 2000.A.D.

3.4 Indian Markets Scenario for Monoclonals Abroad

Many of the scientists contacted in India opine that the use of monoclonal antibodies should be mainly concentrated in the diagnostics are over the next 5-10 years. The major application areas are diagnosis of hepatitis B, filarial parasites and enteric fevers, cancer markers, blood group serology, lymphocyte sub typing, pregnancy and significant animal diseases like FMD, RP, IBR, HS, Brucellosis. The study estimates that around 10 million tests would have to be done every year over the next 10 years for the diagnosis of hepatitis B virus, filiaria parasites to screen and monitor the therapy for such carriers.

Research work pertaining to development of mAbs for diagnosis of T.B., leprosy and other parasites, cancer markers and viruses (example rotavirus) are being undertaken in some of the research laboratories in the country. This would take a few years to materialize. The current and projected potential requirement of monoclonal antibodies is presented I Tables 5.5 and 5.6.                                                                                                                                                                    Back


 

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.

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5.0 Strategic Policy Options

In order to implement the technological alternatives suggested earlier for construction of cell lines and mass production of mAbs, we need to examine some strategic policy options. They are presented below:

Establish a closer linkage between hybridoma laboratories in research institutes, hospitals/clinics and Govt. approved regulatory organizations.

Create a product development cell within established research institutes (having hybridoma labs) to transfer the technology to biotech firms in the industry.

Develop proprietary technology rights and patent rights in order to encourage competitive advantage in organizations.

Fund applied research that would evolve a comprehensive research strategy for the development and transfer of the technology to biotech firms in the pharmaceutical industry.

Canalize imports of raw materials (fetal calf serum) and plastic ware, through central body or institution who will ensure adequate stocks of quality materials acquired at a special price.

Encourage indigenization of technology or import of technology with subsequent indigenization in the area of bio process engineering, raw materials and plastic ware manufacture.

Fund basic research to develop human monoclonals for use in therapy and diagnosis and to overcome technical problems faced in hybridoma technology.

Set up joint collaboration between research laboratories, pharmaceutical companies and Government for upscaling and updating technology especially in mass scale production of monoclonals.

Develop skills of scientists and technicians through a comprehensive training programme at well established research centre.

Foster scientific interaction among clinicians, pathologists, biochemicals and immunologist. This interaction would be necessary to indicate in which are mAbs should be developed and to identify research laboratories that can handle such projects.

Reduce import duties on raw materials and plastic ware used for development and production of diagnostic kits with a condition that supplies to Government hospital for mass screening be charge a special discount price.

Draw up a regulatory framework within which laboratories would be required to enforce strict safety and quality guidelines starting with raw materials used to development of final end product for commercial use in hospitals, clinics and dispensaries around the country.

6.0 Evaluation of the technological options

6.1 Impacts Associated With Hybridoma Technology

An immediate gain will be the replacement of variable and sometime scarce animal antisera. The impacts will be better standardized immunoassays and eventually more economical products.

Rapid diagnosis and subsequent early treatment in the clinic is therefore one of the significant impacts of the hybridoma technology. Rapid diagnosis will enable early treatment thereby saving man hours.

The availability of a large number of specific diagnostic kits will facilitate the task to the general practitioner who will now be able to precisely determine many types of disease, thereby formulating specific therapy without the laborious routine which requires that the patient first be referred to a specialist and then to a pathological laboratory.

The shift in emphasis from therapy (hospitals and drugs) to mass-diffused diagnosis (diagnostic kits) and prevention (vaccines) would lead to reduction in public health costs. According to Prof. Johri, Molecular Biologist at TIFR Bombay, the health costs will rise initially but subsequently decline.

The rapidity, specificity, sensitivity to small quantities of test material (blood, urine), ease of use and low costs will enable the new diagnostic kits to conduct periodic check-ups of individuals and their use can be extended to the entire population.

The hybridoma technology could substantially reduce the cost of developing diagnostic kits if laboratories within the country apply themselves to the task of raising mAbs against specific antigens (especially pathogenic organisms).

The overall impact of early and specific diagnosis coupled with an effective monitoring of therapy will greatly improve the quality of life and have substantial, indirect macroeconomic as well as private economic benefits due to reduction in the number of working days lost through sick leave and the likely prolongation of working life. Improvements in new health-related diagnostics, particularly by monoclonal antibodies which are faster, safer more precise and cheaper than conventional tests, represent a significant improvement in service. The conventional widal test to detect typhoid fever costs around Rs. 30-40/-. However, this test is positive only after seven days of fever and lacks specificity. Moreover, because of high endermicity of the disease, many false positives are also diagnosed.

The diffusion of mass health screening would lead to automation of instrumentation, development of data processing systems and use of telecommunications for transmission of data from laboratories to data processing centers.

The monoclonal antibody technology will have its earliest and greatest impact on the pharmaceutical and health care industry. The market in India by 1991 for monoclonal diagnostic kits is estimated around 100 crores of rupees. This requirement is based on the need for monoclonals to be produced to detect hepatitis B virus, filarion parasites, enteric fevers, pregnancy and cancer.                                                                                                                                                               Back


 

6.2 Impacts associated with the r-DNA technology

New vaccines base don r-DNA technology will be safer than traditional vaccines both for users and producers. Therefore with their increasing numbers and needs for large scale vaccinations, this development will have a major impact in human and veterinary medicine.

There are a number of diagnostic kits based on monoclonal antibodies prepared for diagnosing plant infections whether it is bacteria induced (example crown gall disease, xanthomonas disease of citrus and sorghum) of fungal (e.g. Fusarium on turf grass) or viral.

Human monoclonal antibodies produced by r-DNA technology are desirable and are much more suitable than conventional murine monoclonals for use in therapy and diagnosis. Human mAbs have important applications in oncology, pseudomonas and tetanus infections and autoimmune disease.

The major uses of the r-DNA technology in parasitic diseases are:

1) Specific DNA probes
2) Generation of cloned antigens for immuno diagnosis.
3) Production of cloned antigens for vaccination studies.
4) Characterization of functional protein for drug discovery.

The impact of this technology will also be felt in immunopurification of biologicals and in mass production of mAbs for diagnostic applications.

6.3 Impacts associated with the combination of hybridoma and r-DNA technology

Gen Pharma, a U.S. based biotech firm is trying to use homologous recombination in hybridomas to develop transgenic mice containing complex of genes that produce human immunologists. If successful, the effort should produce mice which makes human mAbs (Biotech news, May 18,1990).

Another significant research development is the production of mAbs from transgenic plants and seeds at 25 cents a gm. (biotech news, April 27, 1990). These developments which combine the hybridoma technology and the r-DNA technology are significant and would encourage the mass production of mAbs at an economical cost. This will also encourage the isolation of therapeutic antibodies and its mass scale production at a relatively inexpensive level. Therefore the impacts are likely to be significant once these technologies are commercialized which is quite likely by 995.

Another related development could be the injection of antibody producing genes into plasmids for transfer to E.Coli and their subsequent production in mass culture systems. Impact of this development could be as significant as the transgenic animal development and is likely to be achieved much earlier.

6.4 Assessment of the technological options

There are three technological options available to India for constructing cell lines to produce monoclonal antibodies. The most popular option is the hybridoma technology option with is being used on a large scale in many research institutes all over the country. A great deal of success has been achieved. However, a few technical problems exist with the technology (for example low frequency of fusion, weak antigens/ high screening load, isotype selection) but feasible improvements in the technology are available to surmount these problems. This calls for a comprehensive approach and additional investment in:

i) Setting up a suitable animal house where the right strains are bred and selected for immunization
ii) Using FACS for early cloning
iii) Working with better myeloma lines
iv) Using electrofusion wherever possible
v) Enriching target lymphocytes before fusion

The earlier section has indicated that the hybridoma technology will have significant impacts at comparatively lesser costs ad investments. This option is therefore a feasible and workable one.

The r-DNA technology option on the other hand is an option that has not been tried in this country for the production of mAbs. Results obtained abroad are still under evaluation. Therefore it would be too early to comment on the feasibility of this option in terms of economic and commercialization aspects. This option will have major impacts in genetically engineering animal mAbs for therapeutic purposes and in immunopurification of biologicals. The demand potential for such applications will be very significant in 2000 A.D. A potential of around 50 crores of rupees is estimated in 2000 A.D. for anti-cancer therapeutics.

The combination of the hybridoma technology and the r-DNA technology option would be very effective for mass scale production. However, sufficient amount of research and development work will be needed to engineer the antibody producing gene in plant or bacterial systems. Leading agricultural universities or institutes doing extensive work in microbial technology would need to be identified to do this kind of research. The impact of this development will be very significant in lowering the cost of diagnostic kits and in producing human mAbs.

Several technologies are currently in use all over the world for large scale production of monoclonal antibodies. The ascites technology is adequate for production of small (gram) quantities of antibody but becomes less appropriate with increase in scale.

Biotech, U.S.A claims that the micro encapsulation technology will be less expensive than the ascites option. Besides, it produces higher concentration of antibodies and does not require maintenance of animals, therefore reduces cost. Moreover, batch-tot-batch reproducibility is obtained which can not be achieved in ascites technology, contamination by virus or immunoglobulin is not obtained as no animal is used in this technology. Ease of purification following culture via micro encapsulation also contrasts favourably with those required by the other large scale manufacturing technologies.

It is also seen that product purity and activity consistent with regulatory requirements is easily achieved. The next best reactor system is the high density hollow fiber perfusion reactor system that has demonstrated a satisfactory yield of mAbs at a cheap cost of production.

The rankings and priorities of strategies for short terms and long term in the case of cell creation and mass production of MAbs is given below in Table E. and E.2.                                                                                                                 Back



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


 

7.3 Identification of R&D centers to transfer the developed technologies to industry

The following institutions could be entrusted the responsibility for producing monoclonal antibodies to be subsequently transferred to the pharmaceutical / biotech industry for packaging and sale.

1) Cancer Research Institute, Bombay: Monoclonals against cancer markers CEA, AFP.
2) NII, New Delhi: Monoclonals against enteric fevers, pregnancy, hepatitis B virus and blood group antigens.
3) Cancer Research Institute, Madras: Monoclonals against cancer markers AFP.
4) Hinduja Hospital and Medical Research Centre, Bombay: Monoclonals against haemopoietic lineage associated markers and thyroid antigens.
5) BAIF Development Research Foundation, Pune: Monoclonals against FMD, RP, IBR
6) MKU University, Madurai: MAbs to detect leprosy.
7) P.G. Institute of Biomedical Sciences, Madras: Monoclonals against hepatitis B virus.
8) TIFR, Bombay: MAbs for large scale therapeutic use with the aid of r-DNA technology.
9) ICGEB, Delhi and CCMB, Hyderabad to develop transgenic plants and human monoclonals.

IMTECH, Chandigarh and NCL, Pune can be asked to develop bioprocess engineering technologies and mAbs for industrial purification. NII, Delhi should be assigned the task of doing basic research in hybridoma technology in order to overcome the technical problems associated with the technology.

BAIF Development Research Foundation, Pune may be considered for taking up large scale production of monoclonal antibodies in their Animal Health Division where fermention and laboratory space with back-up equipments are available. Besides the expertise and learning experience in handling cell lines can be exploited to a tremendous advantage.

7.4 Policies to promote the implementability of the preferred options

(i) Import, technology transfer and joint ventures

Government should encourage imports of patented ENCAPSEL or Hollow fiber continuous flow mass culture technologies and also reduce import duties on encapsels/hollow fibers. This will facilitate large scale production of monoclonal antibodies and r-DNA products. Technology transfer from research centers in India need to be encouraged. This can be done through venture financing (low interest rates on loans), special tax concessions, reduction in import duties on equipment, raw materials and plastic ware, increase in duties on imports of diagnostic kits, and canalization of imports of raw materials (fetal calf serum) and plastic ware through a government agency. Joint ventures with existing companies aboard for particular products or process can also be considered.

(ii) Internal R&D

Government would need to fund basic research to i) develop human monoclonals for use in therapy ii) produce transgenic plants for large scale production of monoclonals iii) develop efficient, indigenous bioprocess engineering technologies and iv) overcome problems faced in existing hybridoma technology. For this a yearly investment of 5 crores of rupees need to be set aside by the government. Secondly, government funded R&D units should be allowed to patent the technology developed. These proprietary technologies can be transferred to industry at a transfer fee and some royalty on sales. Income so obtained can then be used to update the technologies. This would encourage closer links with industry.

Government may also fund applied research. Such research institutions would need to evolve a comprehensive research strategy for development and transfer of technology to biotech firms in the industry. However, it would be desirable if such research is sponsored by firms in the industry and strengthened by close interaction with technical and marketing consultants who will provide marketing, research and technology evaluation inputs. Scientific researchers assigned the projects should be given complete control of projects and finances.

(iii) Regulatory frame work

The government should draw up a regulatory framework and appoint agencies which will have representation from research institutions, users and industry to enforce strict safety and quality guidelines at all levels of development and manufacture of mAbs (raw materials, clinical trials, scaling up, quality control). Safety guidelines should be linked to the updation of the laboratories and penalties need to be raised b the inspecting authority.

(iv) Inter-organizational linkage

It will be necessary to establish closer links between hybridoma laboratories, hospitals/clinics and government approved regulatory organizations to ensure product development, commercialization and its continuous improvement. Joint collaborations between laboratories and industry would need to be set up for the purpose of up scaling and updating technologies developed by such centers. It would also be necessary for research institutions involved in basic and applied research to define and strengthen relationships with international research institutions by publishing research papers aboard, inviting scientists for seminars and workshops and making periodic visits to key research institutions abroad. There is a strong need to encourage scientific interaction among clinicians, pathologists, biochemists and immunologists.

In order to avoid duplication of work at various research centres in India and also to facilitate a kind of coordinative and co-operative mechanism, a study needs to be commissioned by TIFAC or DBT aimed at examining and compiling data on the ongoing work at all te active research centres in the field of veterinary and human disease diagnostics. It is also necessary that an expert committee be formed to resolve issues of cooperation and coordination inn this highly specialized field.

(v) Development of supporting ancillary industry

The ancillary industries need to be geared up to supply packaging material, plastic ware (for example polystyrene ELISA plates, micropipettes) and fetal calf serum. Special tax incentives should be provided by the government to such entrepreneurs involved in providing support services to the biotech firms producing monoclonal kits at an economical price.

(vi) Development of human resources

It would be necessary for some established research institutes like MKU, Madurai, NII, Delhi, CRI, Madras and Bombay to conduct comprehensive training programmes in order to equip technicians and scientists with skills that would enable themselves to confidently handle any problems concerned with the development of monoclonal antibodies and preparation of the diagnostic kit. Training would also be required for medical personnel at primary health centres and clinics/hospitals in order to perform the ELISA test and thereby use the diagnostic kit at their respective units. ELISA READER, microscope and reference album will have to be provided to these centres to facilitate ease of use of the kits.

(vii) Role of financial institutions

Financial institutions should come forward with major equity participation in financing of risk capital projects to enable scientists entrepreneurs to set up biotech firms with indigenous technology for producing diagnostic kits. Long term loans could also be provided at a concessional rate of interest. Equity of financial institutions could be diluted after the project realizes its full economic potential. Any gains that accrue from such an association could be reinvested in centers that would promote utilization of indigenous technology or in providing funds to research institutes for updating technologies.

(viii) Role of pharmaceutical industry

Firms in he industry should have its own R&D and/or would need to sponsor R&D in research laboratories on a continuing basis. Technologies development or acquired need to be productionised by industry in close collaboration with research and regulatory organizations and end users.

(ix) Role of professional and trade associations

Biotech seminars and trade exhibitions can be organized every year to bridge the gap between laboratory, industry end users (clinics, dispensaries) and ventures financiers. Association of the pharmaceutical industry could help in drawing up safety and quality guidelines and in providing a representation to a government cum industry sponsored agency.


 


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