Production and Purification of Monoclonal antibodies by Hybridoma Technology

 G. Kohler and C. Milstein in 1975 first discovered the hybridoma technology. It is used for producing hybrid cells by fusing B-lymphocyte with tumour or myeloma cells. The hybrid cells produced using hybridoma technology, are either cultured in laboratory or sub-cultured using mouse peritoneal cavity. Thus, due to the presence of B-lymphocyte genetic material the hybrid cells can produce antibodies. The tumour cells used to produce hybrid cells make them undergo indefinite division in the culture.

The B-lymphocytes involved are pre-programmed to respond to a single type of antigen or antigenic determinant, thus they produce a single type of antibody that shows specificity for a specific antigen. The reaction of an antigen with B-lymphocyte receptors triggers the rapid division of lymphocytes. As a result, a clone of B cells is produced that generates antibodies against that specific antigen.

This entire process is called clonal selection in which B-lymphocytes produce a single type of antibodies specific to a single type of antigen or antigenic determinant. However, a fully differentiated antibody producing B-lymphocytes (known as plasma cells) does not undergo division when cultured in a laboratory.

Principle

The myeloma cells used in hybridoma technology should not synthesise their own antibodies. The hybridoma cells are selected based on inhibiting the nucleotide (subsequently, the DNA) synthesising machinery. The mammalian cells can synthesise nucleotides either by De novo synthesis or salvage pathway.

Pathways for the Nucleotide Synthesis (HGPRT-Hypoxanthine Guanine Phosphoribosyl Transferase; TK-Thymidine Kinase)

In the De novo synthesis of nucleotides, tetrahydrofolate formed from dihydrofolate is required. Aminopterin (an inhibitor) isolated formed from formation of tetrahydrofolate (and therefore nucleotides)

In the salvage pathway, the purines and pyrimidines are converted into the corresponding nucleotides. The key enzyme involved in the salvage pathway of purines is the Hypoxanthine Guanine Phosphoribosyl transferase (HGPRT) which converts hypoxanthine and guanine to inosine monophosphate and guanosine monophosphate, respectively. Thymidine Kinase (TK) is involved in the salvage pathway of pyrimidines and converts thymidine to Thymidine Monophosphate (TMP). If any of these enzymes (HGPRT or TK) undergo mutation, the salvage pathway gets blocked.

When the mutated cells (i.e., the cells deficient in HGPRT) are grown in a medium containing Hypoxanthine Aminopterin and Thymidine (HAT medium). they fail to survive as the de novo synthesis of purine nucleotides is inhibited. Thus, the cells deficient in HGPRT and grown in HAT medium die.

The hybridoma cells possess the ability of myeloma cells to grow in vitro with a functional HGPRT gene obtained from the lymphocytes fused with myeloma cells. Thus, only the hybridoma cells can proliferate in the HAT medium, and this procedure is used for their selection.

Production of MAbs

The establishment of hybridomas and production of MAbs involves the following steps:
1) Immunisation,
2) Cell fusion,
3) Selection of hybridomas,
4) Screening the products,
5) Cloning and propagation, and
6) Characterisation and storage.

1] Immunisation

The first step in hybridoma technology is to immunise a mouse with a suitable antigen. The antigen and an adjuvant (like Freund's complete or incomplete adjuvant) are injected via subcutaneous route (adjuvants are non-specific potentiators of specific immune responses). The injections are repeated multiple times at many sites.

This increases the stimulation of B-lymphocytes which are responding to the antigen. Three days before the animal is slayed, a final dose of antigen is given via intravenous route. This approach gives rise to large number of immune-stimulated cells for synthesis of antibodies. The concentration of desired antibodies is assayed in the animal serum at frequent intervals during immunisation.

The animal is sacrificed when the concentration of antibodies in serum becomes optimal. The spleen is removed aseptically and disrupted mechanically or enzymatically to release the cells. The spleen lymphocytes are separated from the remaining cells by density gradient centrifugation.

Protocol for the Derivation of Monoclonal Antibodies from Hybrid Myelomas

2] Cell Fusion

The lymphocytes are thoroughly washed and mixed with HGPRT defective imyeloma cells. The mixture of cells is treated with Polyethylene Glycol (PEG) but for a few minutes due to its toxicity. The cells are then washed to remove PEG and kept in a fresh medium. These cells contain a mixture of hybridomas (fused cells), free myeloma cells, and free lymphocytes.

3] Selection of Hybridomas

On culturing the cells in HAT medium, the hybridoma cells grow and the remaining cells disappear slowly within 7-10 days. Selecting a single antibody producing hybrid cells is very essential, and is possible if the hybridomas are isolated and grown individually. The suspension of hybridoma cells is diluted to such intensity that the individual aliquots contain one cell each. These cells are grown in a regular culture medium to produce the desired antibody.

4] Screening the Products

The hybridomas should be screened for the secretion of the antibody of desired specificity, and the culture medium from each hybridoma culture should be tested occasionally (using ELISA and RIA techniques) for the desired antibody specificity. In both the techniques, i.e., ELISA and RIA, the antibody binds to the specific antigen (coated to plastic plates) and the unbound antibody and other components of the medium are washed off. Thus, the hybridoma cells producing the desired antibody are identified by screening. The antibody secreted by the hybrid cells is the monoclonal antibody.

5] Cloning and Propagation

The single hybrid cells producing the desired antibody are isolated and cloned by using the following two techniques:

1. Limiting Dilution Method: In this method, the suspension of hybridoma cells is serially diluted and aliquots of each dilution are transferred into microculture wells. The dilutions are made such that each aliquot in a well contains a single hybrid cell, thus ensuring that the antibody produced is monoclonal.
2. Soft Agar Method: In this method, the hybridoma cells are cultured in soft agar. Many cells can be grown simultaneously in a semisolid medium to form monoclonal colonies.

Practically, both these methods are used in combination to produce maximal MAbs.

6] Characterisation and Storage

The obtained monoclonal antibodies are subjected to biochemical and biophysical characterisation for the desired specificity. The MAbs should also be elucidated for the immunoglobulin class or sub-class, its specific epitope, and its number of binding sites.

The stability of the cell lines and the MAbs is also important. Both are characterised to check their ability to withstand freezing and thawing by freezing the desired cell lines in liquid nitrogen at several stages of cloning and culture.

Large Scale Production of MAbs

Production of MAbs in culture bottles is low (5-10µg/ml), thus to increase the yield the hybrid cells are grown as ascites in the peritoneal cavity of mice. The ascitic fluid contains 5-20mg of MAb/ml, which is much superior to the in vitro cultivation techniques. Collection of MAbs from ascitic fluid has a heavy risk of contamination by pathogenic organisms of the animal. Also many animals have to be sacrificed to produce MAbs. Therefore, workers prefer in vitro techniques over using animals.

Encapsulated Hybridoma Cells for Commercial Production of MAbs

To increase the hybridoma cell density in suspension culture, the hybridomas are encapsulated in alginate gels using a coating solution containing poly-lysine. These gels allow the nutrients to enter in and antibodies to come out of the hybridomas. In this way, the yield of MAb production can be increased (10-100µg/ml).

Purification of MAbs

   The desired antibodies should be extracted from a media sample of cultured hybridomas or a sample of ascites fluid. The contaminants in the cell culture sample consists of growth factors, hormones, and transferrins. The in vivo sample however, contains host antibodies, proteases, nucleases, nucleic acids, and viruses. Other secretions by the hybridomas (like cytokines) may be present in both the cases. Endotoxins may also be present in case of bacterial contamination as they are secreted by the bacteria.

   For purification, the sample is conditioned by removing cells, cell debris, lipids, and clotted materials through centrifugation and then filtration through a 0.45µm filter. Membrane fouling can be caused by the large particles in the further steps of purification. Moreover, the product concentration in the sample might not be sufficient, particularly in cases where the desired antibody is produced by a low-secreting cell line. Therefore, the sample is subjected to ultrafiltration or dialysis for condensation.

   The charged impurities are mostly anions like nucleic acids and endotoxins, and are separated by ion exchange chromatography. At sufficiently low pH, cation exchange chromatography is conducted so that the desired antibody binds to the column and the anions flow through. While at sufficiently high pH, anion exchange chromatography is conducted so that the desired antibody flows through column and the anions bind to it.

Based on their Isoelectric point (pl), many proteins and anions can be separated For example, pI of albumin (4.8) is lower than the pl. of most monoclonal bodies (6.1). So, the average charge of albumin molecules at a certain pH a e negative. Size exclusion chromatography is used for removing transferrin.

Affinity purification is conducted to obtain maximum purity in a single step as the antigen used delivers antibody specificity. In this technique, the antibody generating antigen is covalently bonded to agarose support. If the antigen in a peptide, it is synthesised with a terminal cysteine that allows selective attachment a carrier protein (e.g., Keyhole Limpet Hemocyanin, KLH) during development and to promote purification. The media, containing antibody is incubated with the immobilised antigen in batch. It can also be incubated as the antibody is passed through a column, at which it binds selectively and the impurities are rinsed. The purified antibody is recovered from the support by an elution using a low pH buffer or a gentle high salt elution buffer.

Sodium or ammonium sulphate is used to precipitate out the antibodies for their further selection. Antibodies precipitate at low salt concentrations, while other proteins precipitate at higher concentrations. To obtain best separation, salt should be added in sufficient amount, and the excess salt can be removed by desalting method like dialysis.

Chromatogram is used for the analysis of final purity. Presence of any impurities can be detected by the formation of peaks and the amount of impurity is indicated by the volume under the peaks. Gel electrophoresis and capillary electrophoresis can also be performed for the analysis of final purity. Presence of any impurities can be detected by the formation of bands of varying intensity.