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Ways to produce Monoclonal Antibodies
Our immune system acts as the protective shield of our body against various infections by bacteria and virus causing various diseases. White blood cells, the army of immune system is composed of neutrophils, eosinophil, basophil, lymphocyte and monocyte each carrying out its unique function in fighting against the foreigner entering the body. The B cells of the lymphocytes are the intelligent soldiers which recognize the type of foreign object (antigen) entering the body and releases a weapon called antibody to track the foreigner and destroy them. The two novel traits of an antibody are its specificity to the antigen and once induced its assurance to the body to provide continual resistance to the particular type of disease acquired. Baffled by these two unique features of an antibody, scientists decided to use them for the welfare of the human kind and developed techniques to produce antibodies in vitro. The result is the production of ‘Monoclonal Antibody’.

Monoclonal antibody is the term used for the antibody produced in vitro by multiplying a single hybrid cell, obtained by cloning selected cells from a single source. Monoclonal antibodies are known for its purity and specificity. The conventional method of monoclonal antibody production was done by injecting the test animal with a particular type of antigen. After few days of the dose of the antigen, blood is drawn from the test animal and the antibodies were extracted from the serum of the blood. This method was failure both qualitatively and quantitatively. The antibodies obtained were found to be impure (mixed variants) and the amount obtained was also significantly less. Hence adoption of cloning technique was identified as the optional method to produce antibodies in vitro.

In this method, scientists selected tumor cells for its ability to multiply intensively and the antibody producing mammalian cells and fused these two under in vitro conditions. On the onset of the production, the test animal usually a mice is injected with an antigen to stimulate the antibody production. The antibody producing cells are identified and extracted from the spleen of the mice and it is fused with the myeloma cells which were extracted from the mice earlier and cultured in vitro. The resulting hybrid cell or hybridoma is observed for the presence of the desired antibody and once satisfied, the hybrids are subjected to grow in culture to produce splendid quantity of monoclonal antibodies. Again, the extraction and purification of the monoclonal antibody from the hybridoma is done by sequence of processes like centrifugation, filtration, ultra filtration or dialysis and ion exchange chromatography. Later the ion exchange chromatography was replaced by size exclusion chromatography which was found to be more effective in purifying. Also a procedure called affinity purification was employed to obtain the maximum purity. After undergoing the steps of purification, the final product, the monoclonal antibody is checked for the level of purity by using either chromatogram or gel electrophoresis or capillary electrophoresis.

The first test animal used for the production of monoclonal antibody is mice and a consequence reaction like allergy was observed in humans when supplemented with the monoclonal antibodies produced from mouse cell. Also, humans responded only to the initial dose and developed resistance to further doses. This posed as a bigger problem in obtaining the benefits of the monoclonal antibody and as a result evolved the chimeric antibody. The chimeric antibody is developed by inserting some human amino acid sequence into the animal developed monoclonal antibody.

The novel idea of developing fully human monoclonal antibody is a major breakthrough in the production of monoclonal antibody. In this method, blood sample is collected from an individual (donor) recovered from a particular type of infection and the antibody specific cells are extracted and immortalized. These cells are then subjected to micro well assay technique and the antigen specific antibodies are identified by fluorescence method and isolated. These cells are expanded and characterized before passing to other cell types for large scale production. The difficulty in identifying a donor is eluded by extracting cell from a healthy person and activating the cell for specific antibody production in vitro. The advancement in genetic engineering technology serves the human monoclonal antibody production by using transgenic mice.

The wide therapeutic application of monoclonal antibodies states the significance of the production of the monoclonal antibody.
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Monoclonal antibodies (mAb or moAb) are monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell, in contrast to polyclonal antibodies which are made from several different immune cells. Monoclonal antibodies have monovalent affinity, in that they bind to the same epitope.

1. The first property is exploited by transferring the cell fusion mixture to a culture medium — called HAT medium because it contains:

the pyrimidine thymidine

The logic:

Unfused myeloma cells cannot grow because they lack HGPRT.
Unfused normal spleen cells cannot grow indefinitely because of their limited life span. However,
Hybridoma cells (produced by successful fusions) are able to grow indefinitely because the spleen cell partner supplies HGPRT and the myeloma partner is immortal.

2. Test the supernatants from each culture to find those producing the desired antibody.

3. Because the original cultures may have been started with more than one hybridoma cell, you must now isolate single cells from each antibody-positive culture and subculture them.

4. Again, test each supernatant for the desired antibodies. Each positive subculture — having been started from a single cell — represents a clone and its antibodies are monoclonal. That is, each culture secretes a single kind of antibody molecule directed against a single determinant on a preselected antigen.

5. Scale up the size of the cultures of the successful clones.
Hybridoma cultures can be maintained indefinitely:

in vitro; that is, in culture vessels. The yield runs from 10-60 µg/ml.
in vivo; i.e., growing in mice. Here the antibody concentration in the serum and other body fluids can reach 1-10 mg/ml. However, animal welfare activists in Europe and in the U.S. are trying to limit the use of mice for the production of monoclonals.
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Specialized methods for monoclonal antibody production: (Decoy Method)

To study the embryonic stem cell and their differentiation pattern, it is very impotent to understand the proteins which are involve in the differentiation and to know that what are the different proteins between differentiated and non differentiated embryonic cells.

There is a method called “Decoy method” of producing the monoclonal antibody. This method will produce the antibody for specific protein which is unique in differentiated and non differentiated embryonic cells. For this method, you need group of naive Balb/c mouse (5-6 numbers) which will be immunized with differentiated embryonic cells in one hind foot-pad (Left) and with Non-differentiated embryonic cells in other side (Right) of hind foot-pad.

Differential immunization of these two types of embryonic cells will neutralize all the protein candidates which are common in these two type of cells and antibody response will be only for unique proteins.

Immunization need to be done at every 3 days with 10000 cells (In Phosphate buffer) of each type and for 0-24 days (9 immunizations). After last immunization, Fusion of lymph node cells (Popliteal) need to be done with sp2/0 (Myeloma) cells as routine monoclonal antibody production protocol.

Note: As immunization is done in foot-pad it is important to have very less volume for immunization. While fusion with myeloma cells, left and right lymph cells need to be fused separately.

Obtained monoclonal antibodies need to be screened on differentiated and non differentiated embryonic cells. This method gives best way to discover embryonic marker and production of antibody for them simultaneously.

This is highly advance method to produce antibodies for embryonic markers and can also be applied to search for the cancer markers. Because the fusion efficiency are low with lymph node cells this techniques in not very popular as a routine method for production of monoclonal antibodies, though it is less time consuming in comparison to routine methods.
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Monoclonal antibodies (mAb or moAb) are monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell.

Given almost any substance, it is possible to produce monoclonal antibodies that specifically bind to that substance; they can then serve to detect or purify that substance. This has become an important tool in biochemistry, molecular biology and medicine. When used as medications, the non-proprietary drug name ends in -mab (see "Nomenclature of monoclonal antibodies").

The idea of a "magic bullet" was first proposed by Paul Ehrlich, who, at the beginning of the 20th century, postulated that, if a compound could be made that selectively targeted against a disease-causing organism, then a toxin for that organism could be delivered along with the agent of selectivity. He and Élie Metchnikoff received the 1908 Nobel Prize for Physiology or Medicine for this work, which led to an effective syphilis treatment by 1910.

In the 1970s, the B-cell cancer multiple myeloma was known, and it was understood that these cancerous B-cells all produce a single type of antibody (a paraprotein). This was used to study the structure of antibodies, but it was not yet possible to produce identical antibodies specific to a given antigen.

Production of monoclonal antibodies involving human–mouse hybrid cells was described by Jerrold Schwaber in 1973 and remains widely cited among those using human-derived hybridomas, but claims to priority have been controversial. A science history paper on the subject gave some credit to Schwaber for inventing a technique that was widely cited, but stopped short of suggesting that he had been cheated. The invention was conceived by George Pieczenik, with John Sedat, Elizabeth Blackburn's husband, as a witness and reduced to practice by Cotton and Milstein, and then by Kohler and Milstein. Georges Köhler, César Milstein, and Niels Kaj Jerne in 1975; who shared the Nobel Prize in Physiology or Medicine in 1984 for the discovery. The key idea was to use a line of myeloma cells that had lost their ability to secrete antibodies, come up with a technique to fuse these cells with healthy antibody-producing B-cells, and be able to select for the successfully fused cells.

In 1988, Greg Winter and his team pioneered the techniques to humanize monoclonal antibodies, removing the reactions that many monoclonal antibodies caused in some patients.
Hybridoma cell production
Further information: Hybridoma technology

Monoclonal antibodies are typically made by fusing myeloma cells with the spleen cells from a mouse that has been immunized with the desired antigen. However, recent advances have allowed the use of rabbit B-cells to form a Rabbit Hybridoma. Polyethylene glycol is used to fuse adjacent plasma membranes, but the success rate is low so a selective medium in which only fused cells can grow is used. This is because myeloma cells have lost the ability to synthesize hypoxanthine-guanine-phosphoribosyl transferase (HGPRT), an enzyme necessary for the salvage synthesis of nucleic acids. The absence of HGPRT is not a problem for these cells unless the de novo purine synthesis pathway is also disrupted. By exposing cells to aminopterin (a folic acid analogue, which inhibits dihydrofolate reductase, DHFR), they are unable to use the de novo pathway and become fully auxotrophic for nucleic acids requiring supplementation to survive.

The selective culture medium is called HAT medium because it contains hypoxanthine, aminopterin, and thymidine. This medium is selective for fused (hybridoma) cells. Unfused myeloma cells cannot grow because they lack HGPRT, and thus cannot replicate their DNA. Unfused spleen cells cannot grow indefinitely because of their limited life span. Only fused hybrid cells, referred to as hybridomas, are able to grow indefinitely in the media because the spleen cell partner supplies HGPRT and the myeloma partner has traits that make it immortal (as it is a cancer cell).

This mixture of cells is then diluted and clones are grown from single parent cells on microtitre wells. The antibodies secreted by the different clones are then assayed for their ability to bind to the antigen (with a test such as ELISA or Antigen Microarray Assay) or immuno-dot blot. The most productive and stable clone is then selected for future use.

The hybridomas can be grown indefinitely in a suitable cell culture medium.They can also be injected into mice (in the peritoneal cavity, surrounding the gut). There, they produce tumors secreting an antibody-rich fluid called ascites fluid.

The medium must be enriched during in-vitro selection to further favour hybridoma growth. This can be achieved by the use of a layer of feeder fibrocyte cells or supplement medium such as briclone. Culture-medium conditioned by macrophages can also be used. Production in cell culture is usually preferred as the ascites technique is painful to the animal. Where alternate techniques exist, this method (ascites) is considered unethical.
Purification of monoclonal antibodies

After obtaining either a media sample of cultured hybridomas or a sample of ascites fluid, the desired antibodies must be extracted. The contaminants in the cell culture sample would consist primarily of media components such as growth factors, hormones, and transferrins. In contrast, the in vivo sample is likely to have host antibodies, proteases, nucleases, nucleic acids, and viruses. In both cases, other secretions by the hybridomas such as cytokines may be present. There may also be bacterial contamination and, as a result, endotoxins that are secreted by the bacteria. Depending on the complexity of the media required in cell culture, and thus the contaminants in question, one method (in vivo or in vitro) may be preferable to the other.

The sample is first conditioned, or prepared for purification. Cells, cell debris, lipids, and clotted material are first removed, typically by centrifugation followed by filtration with a 0.45 µm filter. These large particles can cause a phenomenon called membrane fouling in later purification steps. In addition, the concentration of product in the sample may not be sufficient, especially in cases where the desired antibody is one produced by a low-secreting cell line. The sample is therefore condensed by ultrafiltration or dialysis.

Most of the charged impurities are usually anions such as nucleic acids and endotoxins. These are often separated by ion exchange chromatography. Either cation exchange chromatography is used at a low enough pH that the desired antibody binds to the column while anions flow through, or anion exchange chromatography is used at a high enough pH that the desired antibody flows through the column while anions bind to it. Various proteins can also be separated out along with the anions based on their isoelectric point (pI). For example, albumin has a pI of 4.8, which is significantly lower than that of most monoclonal antibodies, which have a pI of 6.1. In other words, at a given pH, the average charge of albumin molecules is likely to be more negative. Transferrin, on the other hand, has a pI of 5.9, so it cannot easily be separated out by this method. A difference in pI of at least 1 is necessary for a good separation.

Transferrin can instead be removed by size exclusion chromatography. The advantage of this purification method is that it is one of the more reliable chromatography techniques. Since we are dealing with proteins, properties such as charge and affinity are not consistent and vary with pH as molecules are protonated and deprotonated, while size stays relatively constant. Nonetheless, it has drawbacks such as low resolution, low capacity and low elution times.

A much quicker, single-step method of separation is Protein A/G affinity chromatography. The antibody selectively binds to Protein A/G, so a high level of purity (generally >80%) is obtained. However, this method may be problematic for antibodies that are easily damaged, as harsh conditions are generally used. A low pH can break the bonds to remove the antibody from the column. In addition to possibly affecting the product, low pH can cause Protein A/G itself to leak off the column and appear in the eluted sample. Gentle elution buffer systems that employ high salt concentrations are also available to avoid exposing sensitive antibodies to low pH. Cost is also an important consideration with this method because immobilized Protein A/G is a more expensive resin.

To achieve maximum purity in a single step, affinity purification can be performed, using the antigen to provide exquisite specificity for the antibody. In this method, the antigen used to generate the antibody is covalently attached to an agarose support. If the antigen is a peptide, it is commonly synthesized with a terminal cysteine, which allows selective attachment to a carrier protein, such as KLH during development and to the support for purification. The antibody-containing media is then incubated with the immobilized antigen, either in batch or as the antibody is passed through a column, where it selectively binds and can be retained while impurities are washed away. An elution with a low pH buffer or a more gentle, high salt elution buffer is then used to recover purified antibody from the support.

To further select for antibodies, the antibodies can be precipitated out using sodium sulfate or ammonium sulfate. Antibodies precipitate at low concentrations of the salt, while most other proteins precipitate at higher concentrations. The appropriate level of salt is added in order to achieve the best separation. Excess salt must then be removed by a desalting method such as dialysis.

The final purity can be analyzed using a chromatogram. Any impurities will produce peaks, and the volume under the peak indicates the amount of the impurity. Alternatively, gel electrophoresis and capillary electrophoresis can be carried out. Impurities will produce bands of varying intensity, depending on how much of the impurity is present.

The production of recombinant monoclonal antibodies involves technologies, referred to as repertoire cloning or phage display/yeast display. Recombinant antibody engineering involves the use of viruses or yeast to create antibodies, rather than mice. These techniques rely on rapid cloning of immunoglobulin gene segments to create libraries of antibodies with slightly different amino acid sequences from which antibodies with desired specificities can be selected. The phage antibody libraries are a variant of the phage antigen libraries first invented by George Pieczenik These techniques can be used to enhance the specificity with which antibodies recognize antigens, their stability in various environmental conditions, their therapeutic efficacy, and their detectability in diagnostic applications. Fermentation chambers have been used to produce these antibodies on a large scale.
Chimeric antibodies
Chimeric antibodies

Early on, a major problem for the therapeutic use of monoclonal antibodies in medicine was that initial methods used to produce them yielded mouse, not human antibodies. While structurally similar, differences between the two are sufficient to invoke an immune response occurred when murine monoclonal antibodies were injected into humans and resulted in their rapid removal from the blood, systemic inflammatory effects, and the production of human anti-mouse antibodies (HAMA).

In an effort to overcome this obstacle, approaches using recombinant DNA have been explored since the late 1980s. In one approach, mouse DNA encoding the binding portion of a monoclonal antibody was merged with human antibody-producing DNA in living cells, and the expression of this chimeric DNA through cell culture yielded partially-mouse, partially-human monoclonal antibody. For this product, the descriptive terms "chimeric" and "humanised" monoclonal antibody have been used to reflect the combination of mouse and human DNA sources used in the recombinant process.
'Fully' human monoclonal antibodies

Ever since the discovery that monoclonal antibodies could be generated in-vitro, scientists have targeted the creation of 'fully' human antibodies to avoid some of the side effects of humanised and chimeric antibodies. Two successful approaches were identified — phage display-generated antibodies and mice genetically engineered to produce more human-like antibodies.

One of the most successful commercial organisations behind therapeutic monoclonal antibodies was Cambridge Antibody Technology (CAT). Scientists at CAT demonstrated that phage display could be used such that variable antibody domains could be expressed on filamentous phage antibodies. This was reported in a key Nature publication.

Other significant publications include:

Marks JD, Hoogenboom HR, Bonnert TP, McCafferty J, Griffiths AD, Winter G (December 1991). "By-passing immunization. Human antibodies from V-gene libraries displayed on phage". J. Mol. Biol. 222 (3): 581–597. doi:10.1016/0022-2836(91)90498-U. PMID 1748994.
Carmen S, Jermutus L (July 2002). "Concepts in antibody phage display". Brief Funct Genomic Proteomic 1 (2): 189–203. doi:10.1093/bfgp/1.2.189. PMID 15239904.

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