How are monoclonal and polyclonal antibodies produced?

Unlike a polyclonal antiserum, a monoclonal antibody (Ab) is a homogeneous preparation specific for a single epitope on the surface of a complex antigen. It was in 1975 that Köhler and Milstein produced the first monoclonal Ab using a method that has quickly become one of the key technologies in immunology.

By fusing activated B cells with myeloma cells, they have been able to form hybrid cells – called hybridomas – which retain the secretory properties of activated B cells and the immortality of myeloma cells. Selected and cloned, these hybridomas constitute a theoretically unlimited source of specific antibodies, all identical. These antibodies first found many applications in basic research and in vitro diagnostics.

Antibody production

In the Clinical Biology laboratory, monoclonal Ab are used as reagents in immunoassays, often replacing traditional antisera. Many years of development and innovation have been necessary to humanize monoclonal Ab, in order to make them usable in human therapy.

Dr. Georges Köhler

Thanks to the development of hybridoma technology in 1975, Georges Köhler, German immunologist, and César Milstein, British biochemist of Argentinian origin, made monoclonal antibodies (Ac) for the first time, which earned them the Nobel Prize in Medicine and Physiology in 1984.

During the years which followed their discovery, monoclonal Ab became revolutionary tools widely exploited in the laboratory, until becoming omnipresent in the field of medical research and diagnosis, as we have explained in detail elsewhere.

In therapeutics, if the monoclonal Ab, known as “first generation”, have been disappointing due to their high immunogenicity and their too short half-life, the progress made subsequently, notably in molecular biology, has made it possible to have much better tolerated molecules.

Thus, other generations of Ac have followed one another: chimeric Acs, then humanized Acs and, finally, totally human Acs, as described in another article of this issue. The applications of these new antibodies are numerous and varied; these include, for example, shrinking or stopping tumor proliferation, inhibiting platelet aggregation, preventing viral invasion, neutralizing toxins, etc.

In this article, we will discuss the methods of obtaining different generations of monoclonal Ab, as well as the comparison of monoclonal Ab with polyclonal antisera, with which they compete in certain in vitro diagnostic applications.

Principle of the production of monoclonal antibodies

In the basic technique, the production of monoclonal Ab takes place in two main stages, namely, the production in vivo (immunization) of lymphoid cells secreting Ab, their hybridization and the in vitro selection of a hybrid producer of Ab , then the multiplication of clones of the hybridoma, either in vitro or in vivo, in order to obtain large amounts of Ac.

Obtaining and selecting hybridomas

The antigen against which we want to prepare Ab is injected into the animal with or without an adjuvant. Booster injections and test bleeds are performed, the latter aimed at verifying that there is production of specific Ac directed against the antigen of interest. Lymphoid cells from the animal’s spleen or lymph nodes are isolated.

They are then fused, in the presence of polyethylene glycol, with myeloma cells previously cultivated and selected in vitro for their deficiency in hypoxanthine-guanine phosphoribosyltransferase (HGPRT–) or, more rarely, in thymidine kinase (TK–). The mixture of all these cells (hybrids and parental cells) is placed on a selective culture medium called HAT medium (Hypoxanthine, Aminopterin and Thymidine).

Selection by the HAT medium depends on the fact that mammalian cells can synthesize nucleotides by two different routes: the de novo route and the recovery route. The de novo pathway, in which a methyl or formyl group is transferred from an activated form of tetrahydrofolate, is blocked by aminopterin, which is an analogue of folic acid.

When the de novo pathway is blocked, cells use the recovery pathway, which bypasses aminopterin blockade by directly incorporating purines and pyrimidines into the nucleotides necessary for the synthesis of DNA and RNA.

Enzymes that catalyze the recovery pathway include HGPRT and TK. A mutation in either of these two enzymes blocks the cell’s ability to use the recovery pathway and causes it to die in the HAT environment.

In hybridoma technology, the myeloma cells used are, in fact, double mutants. As mentioned earlier, they don’t have the HGPRT enzyme. They also lost the ability to produce immunoglobulins (Ig- mutants).

By using Ig- mutants, it is ensured that the Ab produced by the hybridoma are only coded by the spleen cells and that the myeloma cells only bring their property of immortality to the cells resulting from the fusion. The other fusion partner is usually a spleen cell population containing antigen-activated (Ig +) HGPRT + B cells.

These cells contribute to the ability of hybridomas to use the hypoxanthine recovery pathway, thereby enabling their survival in HAT medium. As for unfused B lymphocytes, they disappear after a few days because they are unable to replicate in vitro. Likewise, if hybrids form between B cells or between myeloma cells, they disappear spontaneously.

Monoclonal antibody production

The hybridomas that produce the desired monoclonal Ab are selected and then cloned. We can then multiply the positive clones (producers of the Ac of interest) either by continuing to cultivate them in vitro, or by using the immunized animal for an in vivo production. When a hybridoma is grown in tissue culture flasks, Ac is secreted into the medium, usually at very low concentrations (1-20 µg / mL).

A hybridoma introduced by injection into the peritoneal cavity of a compatible mouse develops there and secretes monoclonal Ab in the ascites fluid at much higher concentrations (usually 1-10 mg / mL approximately). The Ac can then be purified by chromatography of the ascites liquid. To meet the growing demand for monoclonal Ab, in vitro growth techniques for hybridoma cells at very high densities have been developed.

Animals, mice or rats, used both for immunization for the preparation of the hybridoma clone and for the subsequent production of monoclonal Ab (production in vivo), must be of the same strain (syngeneic) for histocompatibility reasons. Balb / c mice are most often used because, in addition, the parental myeloma cells used in the fusion process often come from these mice.

An alternative is to use SCID mice (combined severe immunodeficiency syndrome) which, although expensive, produce less non-specific murine Ab with an equivalent yield of specific monoclonal Ab, which has the effect of facilitating the purification of these.

Monoclonal antibodies for therapeutic use

From 1975, the monoclonal Ab produced by the hybridoma technique are used in therapy and designated by a name ending with the suffix “-momab”. However, these antibodies being of murine origin, the patients treated with these drugs produced anti-mouse antibodies (HAMA, Human Anti-Mouse Antibody).

In addition, these first generation antibodies had a very short half-life after injection and were not able to exercise certain effector functions. These shortcomings therefore led, in 1984, to the production of a new generation of antibodies, chimeric antibodies, designated by the suffix “-ximab” (example of specialty placed on the market: Erbitux®, cetuximab).

In this case, the constant domains of the murine antibody are substituted with the equivalent human constant domains. Consequently, the immunogenic character is reduced but, nevertheless, always remains present.

In order to reduce their immunogenicity, so-called humanized antibodies were developed between 1988 and 1991 and designated by the suffix “-zumab” (example of a specialty placed on the market: Avastin®, bevacizumab).

This third generation of monoclonal Ab consists of human immunoglobulins of which only the hypervariable parts (CDR, Complementary Determining Regions) are of murine origin. This humanization implies, in addition to the permutation of the constant regions, the substitution by human equivalents of the framework regions located within the variable domains.

Most often, this is a CDR graft on the framework regions of a human antibody. The method used consists of a guided selection based on surface presentation techniques of bacteriophages.

Finally, from 1994 to 1999, a last generation of antibodies was born, completely human antibodies, with the suffix “-mumab” (example of specialty placed on the market: Vectibix®, panitumumab).

Obtaining human hybridomas is very delicate, other methodologies are used to create these antibodies of human origin: humanized transgenic mice (human antibody genes are introduced in place of the mouse’s immune genetic material) or the combinatorial banks of antibodies expressed on the surface of bacteriophages (phage display).

In addition, in certain situations, the use of antibody fragments (Fab – antigenbinding Fragment, scFv – single-chain Fragment variable) has proven to be more relevant. Indeed, the presence of Fc receptors is not always required: this is the case, for example, during cytokine inactivation or viral neutralization.

In addition, these fragments being smaller than the whole antibody, they may have better bioavailability and be more easily produced in prokaryotic systems or yeasts.

Production of polyclonal antibodies

Polyclonal Ab are Ab produced by several families of B lymphocytes. Consequently, they recognize several epitopes, including on the same antigen. Antisera containing these polyclonal Ab are usually produced by injecting a target immunogen (antigen) into an animal, often combined with an adjuvant that enhances the immune response.

The latter can then be amplified by booster injections of antigen with or without adjuvant. Blood samples are taken from the animal to assess the level of Ac production. When the titer is high enough, the antiserum is prepared by performing a large blood test followed by isolation of the serum and, if necessary, purification of the Ac from the serum.

The choice of animal species used depends on several factors, including the volume of serum required (which itself depends on the amount of polyclonal Ab to be produced) and the type of immunoassay. The age, gender and health of the animal are also important.

The most commonly used animal species for the production of polyclonal CAs are rabbits, mice, rats, hamsters, guinea pigs, goats, sheep and chicken. Large animal species (eg horses) can also be used to obtain larger volumes of antiserum, but maintaining these animals is more costly.

In addition, immunoglobulins can be extracted from the milk of cattle, sheep and goats, which is a non-invasive method for producing large volumes of polyclonal Ab.

What are the advantages and disadvantages of monoclonal antibodies?

For precise targeting as required in therapeutic applications, the use of monoclonal Ab is essential. Regarding the use as reagents in in vitro diagnostics, polyclonal antisera still retain interest in certain assay techniques.

Indeed, polyclonal Ab can be obtained much more quickly, cheaply and with less technical skills than what is required to produce monoclonal Ab. It is possible to obtain polyclonal Ab in 4 to 8 weeks, while production of monoclonal Ab can take 3 to 6 months. However, this drawback must be weighed against the availability of monoclonal Ab once the hybridomas have been immortalized.

In addition, the production of monoclonal Ab requires fewer animals compared to the production of polyclonal Ab, but the production of the former by the ascites method causes discomfort and pain in animals. Given the diversity of antibodies secreted in the polyclonal response, polyclonal antisera are difficult to reproduce.

The quantity and quality of antibodies produced varies from animal to animal and even from one animal to another at different times. Consequently, the availability of polyclonal Ab is limited, and their characteristics may change during the production period. In addition, the quantity of polyclonal Ab obtained is limited by the size of the animal and by its lifespan.

On the other hand, monoclonal Ab, being from the same clone, are specific for the same epitope for which they have a determined affinity. They are produced constantly over time and in unlimited quantities. Indeed, hybridomas are kept for years in in vitro culture without modification of the Ac they produce and, moreover, these cells can be frozen.

However, as the monoclonal Ab is directed specifically against a single epitope, it is sufficient that it undergoes a minor modification (denaturation, genetic polymorphism, etc.) for its binding capacity with the antigen to be lost. Conversely, since polyclonal Ab have different affinities for different epitopes and can bind to several different sites of the same complex antigen, a change in one or a small number of epitopes is less likely to alter their functions.

In order to avoid possible problems due to the monospecificity of the monoclonal Ab and to increase their heterogeneity, we sometimes combine two or three of them, but it is often difficult, too expensive and too long to identify multiple monoclonal Ab with desired specificity . Monoclonal Ab obtained in vivo (ascites) or in vitro (culture) have a concentration often 10 times higher than that of polyclonal Ab and a much higher purity.

What should we remember?

Since it has been possible to produce them artificially and in sufficient quantity, monoclonal Ab have found important applications in in vitro diagnostics. The interest of monoclonal Ab in the biomedical analysis laboratory was the subject of a specific article in this same issue.

Monoclonal Ab allowed the development of very sophisticated assay methods that allow quantification of many markers in biological media, with a specificity very significantly improved compared to polyclonal antisera. These are still widely used in certain techniques, due, in particular, to their lower cost.

Thanks to advances in biotechnology and, in particular, to genetic recombination techniques, it is now possible to obtain modified monoclonal Ab that are increasingly close to human immunoglobulins. These Ac, chimeric, humanized or human, present a reduced risk of inducing an immunogenic response in humans.

Thus, currently, these new generations of recombinant monoclonal CAs have become drugs whose considerable clinical potential affects various medical fields, in particular those of oncology and autoimmune diseases.