monoclonal antibody monoclonal antibody a monoclonal antibody (mab, more rarely called moab) is an antibody produced from a cell lineage made by cloning a unique white blood cell. all subsequent antibodies derived this way trace back to a unique parent cell. a general representation of the method used to produce monoclonal antibodies monoclonal antibodies can have monovalent affinity, binding only to the same epitope (the part of an antigen that is recognized by the antibody). in contrast, polyclonal antibodies bind to multiple epitopes and are usually made by several different antibody-secreting plasma cell lineages. bispecific monoclonal antibodies can also be engineered, by increasing the therapeutic targets of one monoclonal antibody to two epitopes. it is possible to produce monoclonal antibodies that specifically bind to almost any suitable substance; they can then serve to detect or purify it. this capability has become an investigative tool in biochemistry, molecular biology, and medicine. monoclonal antibodies are being used on a clinical level for both the diagnosis and therapy of several diseases. in 2020, the administration of monoclonal antibodies was authorized by several countries for treating moderate symptoms of covid-19. history in the early 1900s, immunologist paul ehrlich proposed the idea of a zauberkugel – "magic bullet", conceived of as a compound which selectively targeted a disease-causing organism, and could deliver a toxin for that organism. this underpinned the concept of monoclonal antibodies and monoclonal drug conjugates. ehrlich and élie metchnikoff received the 1908 nobel prize for physiology or medicine for providing the theoretical basis for immunology. by the 1970s, lymphocytes producing a single antibody were known, in the form of multiple myeloma – a cancer affecting b-cells. these abnormal antibodies or paraproteins were used to study the structure of antibodies, but it was not yet possible to produce identical antibodies specific to a given antigen.: 324 in 1973, jerrold schwaber described the production of monoclonal antibodies using human–mouse hybrid cells. this work remains widely cited among those using human-derived hybridomas. in 1975, georges köhler and césar milstein succeeded in making fusions of myeloma cell lines with b cells to create hybridomas that could produce antibodies, specific to known antigens and that were immortalized. they and niels kaj jerne shared the nobel prize in physiology or medicine in 1984 for the discovery. in 1988, gregory winter and his team pioneered the techniques to humanize monoclonal antibodies, eliminating the reactions that many monoclonal antibodies caused in some patients. by the 1990s research was making progress in using monoclonal antibodies therapeutically, and in 2018, james p. allison and tasuku honjo received the nobel prize in physiology or medicine for their discovery of cancer therapy by inhibition of negative immune regulation, using monoclonal antibodies that prevent inhibitory linkages. production looking at slides of cultures of cells that make monoclonal antibodies monoclonal antibodies can be grown in unlimited quantities in flasks. hand-filling wells with a liquid for a research test. this test involves preparation of cultures in which hybrids are grown in large quantities to produce desired antibody. this is effected by fusing a myeloma cell and a mouse lymphocyte to form a hybrid cell (hybridoma). bathing prepared slides in a solution hybridoma development much of the work behind production of monoclonal antibodies is rooted in the production of hybridomas, which involves identifying antigen-specific plasma/plasmablast cells that produce antibodies specific to an antigen of interest and fusing these cells with myeloma cells. rabbit b-cells can be used 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 possible 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. exposing cells to aminopterin (a folic acid analogue, which inhibits dihydrofolate reductase, dhfr), makes them unable to use the de novo pathway and become fully auxotrophic for nucleic acids, thus 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 medium because the spleen cell partner supplies hgprt and the myeloma partner has traits that make it immortal (similar to 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-media conditioned by macrophages can be used. production in cell culture is usually preferred as the ascites technique is painful to the animal. where alternate techniques exist, ascites is considered unethical. novel mab development technology several monoclonal antibody technologies have been developed recently, such as phage display, single b cell culture, single cell amplification from various b cell populations and single plasma cell interrogation technologies. different from traditional hybridoma technology, the newer technologies use molecular biology techniques to amplify the heavy and light chains of the antibody genes by pcr and produce in either bacterial or mammalian systems with recombinant technology. one of the advantages of the new technologies is applicable to multiple animals, such as rabbit, llama, chicken and other common experimental animals in the laboratory. purification after obtaining either a media sample of cultured hybridomas or a sample of ascites fluid, the desired antibodies must be extracted. cell culture sample contaminants 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, one or the other method (in vivo or in vitro) may be preferable. 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 produced by a low-secreting cell line. the sample is therefore concentrated by ultrafiltration or dialysis. most of the charged impurities are usually anions such as nucleic acids and endotoxins. these can be 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 along with the anions based on their isoelectric point (pi). in proteins, the isoelectric point (pi) is defined as the ph at which a protein has no net charge. when the ph > pi, a protein has a net negative charge, and when the ph < pi, a protein has a net positive charge. 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. thus, at a ph between 4.8 and 6.1, the average charge of albumin molecules is likely to be more negative, while mabs molecules are positively charged and hence it is possible to separate them. transferrin, on the other hand, has a pi of 5.9, so it cannot be easily separated 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. this method 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 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 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 support purification. the antibody-containing medium 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. antibody heterogeneity product heterogeneity is common in monoclonal antibodies and other recombinant biological products and is typically introduced either upstream during expression or downstream during manufacturing. these variants are typically aggregates, deamidation products, glycosylation variants, oxidized amino acid side chains, as well as amino and carboxyl terminal amino acid additions. these seemingly minute structural changes can affect preclinical stability and process optimization as well as therapeutic product potency, bioavailability and immunogenicity. the generally accepted purification method of process streams for monoclonal antibodies includes capture of the product target with protein a, elution, acidification to inactivate potential mammalian viruses, followed by ion chromatography, first with anion beads and then with cation beads. displacement chromatography has been used to identify and characterize these often unseen variants in quantities that are suitable for subsequent preclinical evaluation regimens such as animal pharmacokinetic studies. knowledge gained during the preclinical development phase is critical for enhanced product quality understanding and provides a basis for risk management and increased regulatory flexibility. the recent food and drug administration's quality by design initiative attempts to provide guidance on development and to facilitate design of products and processes that maximizes efficacy and safety profile while enhancing product manufacturability. recombinant the production of recombinant monoclonal antibodies involves repertoire cloning, crispr/cas9, or phage display/yeast display technologies. recombinant antibody engineering involves antibody production by the use of viruses or yeast, 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 phage antigen libraries. these techniques can be used to enhance the specificity with which antibodies recognize antigens, their stability in