In recent years, drug discovery researchers have expanded their portfolio of products from small-molecule drugs to large biomolecules such as recombinant proteins or monoclonal antibodies developed for diagnostic or therapeutic uses. The production of these important biomolecules often requires many chromatographic steps to obtain pure products and ensure that no contaminants are present that might cause adverse effects such as allergic reactions or even death.
The amount of purified protein required differs depending on the research and development phase in which the biomolecule of interest is being evaluated. For example, in the early discovery phase, smaller amounts of multiple antibody constructs, each with different binding characteristics, are required for testing and analysis. Methods such as surface plasmon resonance are used to determine the kinetic parameters for the binding of these antibodies with their targets and to allow for the identification of antibodies that have the desired binding characteristics. These antibodies are then further investigated in the preclinical phase. Ultimately, clinical trials are performed to determine the effectiveness of experimental drugs in humans. Once an effective antibody has been identified, the last phase of the drug discovery workflow requires large amounts of purified product. At the large scale, similar separation techniques are utilized but with a much higher throughput.
There are many different methods for antibody production; two of the most popular methods are the creation of antibodies and antibody fragments from mouse hybridoma cell lines, and phage display. Both methods rely on chromatography to isolate and purify antibodies and antibody fragments (Fabs) from complex media, such as cultured mouse cell lines or bacterial supernatants. Because these media contain highly complex protein mixtures, extensive multistep chromatography is often required to produce purified antibody products. This section describes how chromatography can be used in the discovery of monoclonal antibodies and fragments.
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Monoclonal antibodies are typically purified from crude samples derived from either culture media or ascites taken from a host animal. These crude samples contain many contaminants such as growth factors, hormones, DNA, endotoxins, and host cell proteins. A typical workflow for monoclonal antibody purification involves initial centrifugation and filtration steps prior to chromatography. Many chromatographic methods can be employed in antibody purification. A one-step solution utilizes affinity chromatography. Here, the crude sample is passed through a column filled with a resinous stationary phase containing protein A, which captures the antibody because protein A has a high specificity for the Fc (fragment, crystallizable) regions of antibodies (see figure below). Although affinity chromatography is a simple and quick approach, it has the drawback of being expensive compared with other methods. Much of this expense is due to the shorter lifetime of protein A resin compared with other stationary phases. Tagged affinity chromatography is another frequently employed method. In this process, the antibody is tagged with a molecule such as a histidine tag. The column contains a molecule such as tris-NTA that binds the tag with high affinity, enabling the antibody to be captured and purified in one step. However, this approach produces an antibody with a tag attached, and this tag may interfere with the binding of the antibody to its target antigen. This tag must therefore be removed, adding downstream processing to the workflow.
Schematic structure of an antibody. Fab, antigen-binding region; Fc, crystallizable region.
Historically, affinity chromatography is only a part of a multistep approach wherein the crude sample is first passed through an affinity column and subsequently polished using ion exchange chromatography (IEX). Cation exchange is a popular method for cleaning up an isolated monoclonal antibody. In this step, the antibody binds the solid phase, and contaminants are allowed to flow through or wash off the protein. The reverse (anion exchange) method may also be employed, in which case contaminants are captured on the anion exchange column, while the antibody flows through. Two ion-exchange chromatography steps are often needed for the complete removal of cell-related contaminants. Size-exclusion chromatography is a less popular final step for removing residual proteins and contaminants; this method often employs a size-based filter rather than a column due to volume constraints.
Phage display is a common method for the production of antibody fragments (Fabs), so named because these fragments contain only an antigen-binding region (see figure above). Phage display is widely used by researchers in the development of novel therapies or the identification of novel targets for therapies because millions of Fabs can be produced in a single experiment. These Fabs are subjected to an initial screening to identify those with highest affinity for their intended targets. Successfully selected Fabs are often converted into full-length antibodies for possible therapeutic use; however, successful translation into clinical practice remains rare.
Many different chromatographic methods can be employed to purify Fabs from an initial crude cell culture supernatant in a manner similar to that described above for monoclonal antibody purification. One common method is affinity tag chromatography, whereby a tag is expressed as part of the Fab during phage display to allow for easy isolation and purification of the Fab. A histidine tag is commonly used; the method is the same as that described in the previous section.
