In this article we will discuss about the technology used and commercial production of monoclonal antibodies.
Technology Used in Production of Monoclonal Antibodies:
Hybridoma Technology:
To overcome the limitations of the use of antiserum, a need of new technique was long felt by the immunologists, and that dream was realized during the recent past with the help of “somatic hybridization” (animal cell fusion).
Two brilliant immunologists, German born G.F. Koehler and C. Milstein of Argentina developed a new technology, namely, hybridoma technology in 1975, which later (in year 1984) earned Nobel Prize for them in medicine.
Koehler and Milstein demonstrated that somatic hybridization technique could be used to generate continuous “hybridoma cell line (hybridomas)” or the “hybrid cell line” secreting specific antibodies called monoclonal antibodies (Mabs).
What Koehler and Milstein did was to fuse normal antibody-producing mouse B-lymphocytes with mouse-myeloma B-lymphocytes (cancerous B-lymphocytes) to generate hybridoma cells, the fusion product of the two cell lines.
Since the hybridoma cell had been the fusion product of the B-lymphocyte and myeloma B- lymphocyte, it enjoyed the properties of both the partners; the property inherited from normal B-lymphocyte to produce specific antibody, and the property inherited from myeloma (cancerous) B-lymphocyte to multiply uncontrollably and grow indefinitely in culture.
Therefore, the hybridoma cells are capable of growing indefinitely in culture and of secreting specific antibody molecules continuously. As we know that one type of antibody-secreting normal B-lymphocyte secretes a specific type of antibody molecules, all the antibodies secreted in culture by a particular hybridoma cell line remain identical.
Since these antibodies are resulted in by a clone of cells developed from a single hybridoma in culture, they are said to be monoclonal antibodies (Mabs).
Making a Hybridoma:
Following main steps are involved in making a hybridoma and enabling it to successfully grow in culture medium to produce monoclonal antibodies:
(i) Immunization:
An animal, e.g., a mouse, is first immunized with the appropriate antigen. Generally the antigen is injected subcutaneously or into the peritoneal cavity of the animal alongwith an adjuvant to stimulate the immune system. The animal is injected on several occasions and with such immunization, there is increased stimulation of B-lymphocytes which respond to the antigen.
The final dose of the antigen is given intravenously three days before the animal is killed. The intravenous injection ensures a high dose of antigen and, three days after immunization, the immune-stimulated cells will be growing maximally. This simplifies the selection process later.
(ii) Killing of Animal and Separation of Lymphocytes:
Three days after the final dose of antigen has been given intravenously to immunize the animal, the latter is killed. The spleen of the killed animal is aseptically removed and gently disrupted to release the spleen fluid containing lymphocytes and red blood cells. The lymphocytes are separated from the spleen fluid (and red blood cells) by density gradient centrifugation, and are washed.
(iii) Selection of Myeloma Cell Line:
One of the most important point deserving attention during the course of monoclonal antibody production is that just no myeloma lymphocyte be allowed to fuse with normal lymphocyte.
The myeloma cell line used must itself not be capable of synthesizing antibody otherwise hybridoma cell line will produce a mixture of antibodies. It is worth noting that in most cases of multiple myeloma there is overproduction of a particular type of antibody.
Such myeloma cell lines that do not produce antibodies are readily available because there are instances where a particular myeloma cell line multiplies uncontrollably without antibody production. HPRT-negative myeloma cell line is such an example.
(iv) Fusion of Separated Lymphocytes with Myeloma Lymphocytes:
After washing, the lymphocytes are mixed with myeloma lymphocytes of HPRT-negative cell line. The mixture of the two cell lines is now exposed to polyethylene glyclol, a fusion promoting agent.
The latter is cytotoxic and, therefore, the exposure is limited only to a few minutes. The cells are then washed to make them polyethylene glycol free. The washed cells comprise a mixture of hybridoma cells, un-fused lymphocytes and un-fused myeloma lymphocytes.
(v) Selection of Hybridoma Cells:
Since the mixture of normal lymphocytes and myeloma lymphocytes contains hybridoma cells, un-fused normal lymphocytes, arid un-fused myeloma lymphocytes, the hybridoma cells have to be selected from the mixture for further requirements. This is done by using HAT-medium for growth. HAT-medium represents a mixture of hypoxanthine, aminopterin and thymidine.
When the said mixture of cells is grown in the HAT-medium, the un-fused normal lymphocytes and the un-fused myeloma lymphocytes fail to grow. However, the hybridoma cells grow successfully in the HAT-medium because they possess the ability of myeloma lymphocytes to grow in vitro and the normal HPRT gene inherited from normal lymphocytes.
(vi) Isolation of a Monoclonal antibody Producing Hybridoma Cell:
If all the hybridoma cells that has been selected using HAT-medium are grown together, then a polyclonal antibody mixture would be obtained. Consequently, a single antibody (monoclonal antibody) producing hybridoma cells need to be isolated and grown individually.
This is done by diluting a suspension of hybridoma cells to such an extent that individual aliquots contain, on an average, only one cell. Such cells are transferred to separate fresh media for growth.
Each mass of hybridoma cells (clone) produced from a single parent hybridoma cell is now examined to determine whether it produces the desired monoclonal antibody. A schematic representation of various steps involved in making a hybridoma are summarised in Fig. 41.10.
Commercial Production of Monoclonal Antibodies:
Once the correct hybridom has been selected and isolated, it can be stored frozed (cryopreserved) and cultured whenever required. Since the hybridoma is a transformed cell line, it grows readily in culture but the antibody titre is low, say generally 5-10 mg/l. This rate of monoclonal antibody production, which is very low seeing its requirements, has been considerably enhanced using large scale production methods.
1. Microencapsulation Technology:
Microencapsulation technology is an interesting method of large scale production of monoclonal antibodies and has been patented by the Damon Corporation. In this
technology, the hybridoma cells/hybridomas are grown inside hollow microspheres which arc surrounded by a porous membrane.
Protected inside the microsphere, the cells multiply and grow to higher cell densities than is possible with any other culture system. Since the antibody is retained by the membrane, it does not become contaminated with other immunoglobulin’s if serum is added in the growth medium.
This technology results in the production of 100 mg to one g of antibody per litre of culture medium and makes large scale production of monoclonal antibodies (MABs) commercially feasible (Fig. 41.11).
2. Ascites-route Technology:
Ascites-route technology provides an alternative for monoclonal antibody production commercialIy. In this technique, the hybridoma is grown as ascites, i.e., as a suspension of tumour cells within the peritoneal cavity of an isogenic species. The ascitic fluid, which is formed following injection of the tumour, generally contains as much as 19.95 gm/l of antibody.
This amount is superior to that obtained by the best in vitro cultivation technique.
Some commercial suppliers of monoclonal antibodies use the ascites- route technology but this technology has considerable disadvantages:
(i) It is labour intensive
(ii) Specific pathogen-free animals required for use and kept quarantined at all times to prevent contamination of the monoclonal antibodies.
These disadvantages add greatly to the cost of Mabs. In addition, why sacrifice large numbers of laboratory animals when an alternative approach is already available?
3. Mass in Vitro Culture:
Large-scale culture of hybridoma cells has been done in stirred bioreactors, airlift fermenters and in vessels based on immobilized cells. The culture systems using immobilized cells enable the cultivation of cells at very high densities, which markedly enhances the antibody yields.
For example, some hollow fiber cartridges (a culture system), produce upto 40 g Mabs per month, while opticel systems (special ceramic cartridges) may yield about 50 g antibody/day. It is expected that further developments in immobilized culture systems will enhance antibody yields considerably and markedly reduce the cost of their production from cell cultures.
The medium used for in vitro culture of hybridomas must serve the following two purposes:
(1) Support adequate cell proliferation and
(2) Give high antibody yields.
Most basal tissue culture media have been designed for cell proliferation or for production. In general, four basal media (DMEM, IMDM, F12 and RPMI 1640) are used either singly or in combination, and are almost always supplemented with insulin, transferrin (stimulates cell proliferation), ethanolamine, and often with bovine albumin serum.
Many hybridomas require additional hormones, growth and differentiation factors. A 1: 1: 2 combination of DMEM, Ham’s F12 and RPMI 1640 supplemented with amino acids, vitamins, insulin, transferrin, ethanolamine and selenite gave the best results for proliferation of and IgM production from human hybridoma cells. Most likely, the biotechnology companies would have devised several undisclosed media formulations.
It is fairly easy to produce rodent Mabs, but there are various difficulties in the production of human Mabs. Rodent Mabs are good enough for diagnostic and separation procedures, but for therapeutic purposes human Mabs are far more desirable.
Since antibodies are themselves immunogenic (and antigenic), human Mabs are likely to be much better tolerated by patients than rodent Mabs. In addition, human Mabs can be prepared against those antigens as well, which are non-immunogenic in rodents.
The chief problem with the production of human hybridoma clones relates to the collection of lymphocytes producing specific antibodies since human beings can not be immunized at will and their spleen (or lymph node) can not be excised (the normal practices in rodent hybridoma production). Human lymphocytes are usually collected from blood of volunteers who have been immunized with some antigen, e.g., tetanus toxoid.
The antibody producing lymphocytes are then immortalized in one of the following three ways:
(i) The B-lymphocytes arc transfected with DNA from a tumour; this transforms the lymphocytes making them capable of indefinite proliferation.
(ii) Fusion of the B-lymphocytes with suitable lymphoid tumour cells to produce hybridoma clones. The tumour cell line must be antibody nonproducing (Ig–).
(iii) Transformation of B-lymphocytes following their infection with Epstein Barr virus to lymphoblastoid cell lines.
These lymphoblastoid cell lines can either themselves be used for Mab production or may be used to produce hybridoma clones (they are used in place of the B-lymphocytes).
At present, the second and third approaches of producing human-human hybridoma clones are being most commonly used. Several human Mabs are available commercially, e.g., against lung carcinoma, breast carcinoma, tetanus toxoid (human-mouse hybridoma), against glioma, measles virus (human-human hybridoma), against RhD, hepatitis B surface antigen (Eptein-Barr virus transformed B-lymphocytes), and against tetanus toxoid (hybridoma involving fusion of Epstein-Barr induced transformed lymphocytes in place of normal B-lymphocytes).
It has been generally reported that Mabs exhibit lower affinities when compared with the effective affinities of their polyclonal counterparts. But Mabs recognizing peptide antigens with very high affinities (affinity constant up to 1011 mol-1) have been produced, in practice it may be necessary to screen several different Mabs against the same antigen in order to identify a Mab suitable for the given purpose.