cell and a
mouse lymphocyte to form a hybrid cell (
hybridoma).
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.
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) 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 80%) is obtained. The generally harsh conditions of this method may damage easily damaged antibodies. 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 various environmental conditions, their therapeutic efficacy and their detectability in diagnostic applications. Fermentation chambers have been used for large scale antibody production.
Chimeric antibodies While mouse and human antibodies are structurally similar, the differences between them were sufficient to invoke an immune response when
murine monoclonal antibodies were injected into humans, resulting in their rapid removal from the blood, as well as systemic inflammatory effects and the production of
human anti-mouse antibodies (HAMA). Recombinant DNA has been explored since the late 1980s to increase residence times. In one approach called "CDR grafting", mouse DNA encoding the binding portion of a monoclonal antibody was merged with human antibody-producing DNA in living cells. The expression of this "
chimeric" or "humanised" DNA through
cell culture yielded part-mouse, part-human antibodies.
Human antibodies and single B cell cloning. == Cost ==