Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Monoclonal antibodies (mAbs) are essential tools for research scientists in a range of biological and biochemical disciplines. As molecules designed to bind with high specificity to known epitopes, they are in demand for everything from affinity purification to in vivo labeling. Pharmaceutical companies also have an interest in mAbs because these molecules are being hotly pursued as therapeutic drugs against human diseases. While most researchers require relatively small amounts of mAbs, pharmaceutical companies, large research and industrial labs require much larger amounts. But obtaining large quantities of mAbs is time-consuming and expensive using traditional antibody production methods — immunizing small animals and harvesting antibodies after they develop immunity. However, in vitro methods of antibody production have also evolved to be used on a variety of production scales. Here we look at the methods commonly used for large-scale (over 0.5-1 gram) production and purification of mAbs, which is aiming to save researchers and industry both time and money.
Antibody-generating cells
Whether to use in vitro or in vivo methods of large-scale antibody production depends on a variety of factors, such as cost and time spent waiting for production, but also the particular preferences of the researcher. The cost of the two methods has become nearly comparable when all expenses are factored in. In vivo mAb generation generally takes a minimum of about 6 weeks, whereas in vitro production can take longer if additional steps such as optimization of the cell line are needed.
A common method of generating mAbs in vitro is the use of hybridoma cells. These are cultured cell lines that result from the fusion of myeloma cells with spleen cells from an animal immunized with the antigen. Clones grown from these cells are tested to determine whether the antibodies they secrete bind to the antigen of interest. The positive clones are chosen and grown, all the while secreting mAbs into the growth media, which is collected regularly.
If you inject the positive hybridomas into lab animals such as mice, they will develop into tumors that secrete ascites fluid, which is rich in antibodies. Whenever possible, however, it is preferable to produce ascites fluid in cell culture (as described above) for ethical reasons, as it is painful for the animals. However, some hybridoma cell lines simply don’t grow effectively in culture, may take too long to grow, or may not secrete enough antibodies under cell culture conditions, in which case the in vivo method remains an alternative.
Hollow fiber bioreactors
Small scale in vitro mAb production relies on growing hybridomas in flasks, cell bags, or other labware. But for large-scale production, one needs to use larger containers that can hold many more cells and encourage a much higher titer, which is the concentration of antibodies in solution. One common tool is the use of hollow fiber bioreactors, which allow the extremely efficient growth of antibody-secreting cells at high densities, resulting in very high titers. As a rough guideline, they are commonly used for producing mAbs in the range of 1–10 grams.
Several factors contribute to the efficiency of hollow fiber bioreactors, also known as fiber cell systems. The cells grow in a more natural 3-dimensional, porous matrix environment, rather than the traditional 2-dimensions of a typical cell culture dish configuration. In conventional cell culture, quickly growing cells may end up competing for nutrients and space in the medium, which can slow the secretion of antibodies. In contrast, hollow fiber bioreactors usually incorporate a perfusion system that continuously adds fresh growth media and harvests used media (which is precious because it contains the desired mAbs). This also means that the cells experience less toxicity from incubating in their own waste, and in fact can grow up to densities that resemble in vivo tissues, without adverse effects. In fact, cells grown in hollow fiber bioreactors can produce antibody titers up to 100 times higher than cells grown under conventional culture conditions. Additional benefits for the researcher are that hollow fiber bioreactors are reasonably inexpensive (they can even be homemade), spatially compact, and easy to use, and require less maintenance than cells grown under in conventional cell culture conditions.
Stirred tank bioreactors
As a general guideline, stirred tank bioreactors are often used for producing mAbs in amounts greater than 1 gram, though are sometimes reserved for amounts over 10 grams. Typically these bioreactors are very large (holding from 5 to 2000 L) cylindrical tanks that rely on a rotational mixing system on either the top or bottom of the tank. They usually include other mechanisms for additional mixing and/or minimizing gas bubbles, such as marine impellers, turbines, or baffles. These large scale units are easier to use today, because you can purchase single-use units that are already assembled and sterilized. An important benefit of stirred-tank bioreactors is their track record of use by large-scale production facilities in research and biopharmaceutical applications; these facilities have spent time and money optimizing growth and production conditions. Another advantage is the availability of stirred-tank bioreactors that allow fully automated control of the internal environment of the bioreactor.
Purification of mAbs
Regardless of the method used to grow the cells, the antibodies they secrete into the media must be purified before use. Otherwise, the desired antibodies may be mixed with components from the cell culture media, or factors from the host animal if grown in vivo. Both will also contain other substances secreted by the hybridomas that are producing the antibody, such as cytokines. Other contaminants such as bacteria and endotoxins might also be present.
Purification begins with centrifuging and filtering the secreted media to remove larger debris, cells, and cell fragments. Subsequently, chromatography (such as ion exchange or size exclusion) can help to rid the sample of unwanted anions, such as nucleic acids or transferrin, for example. Alternatively, protein A/G chromatography can separate out the desired antibodies by binding them, while everything else flows out the column. While this method is quick, with high purity, the low pH used for elution may damage the antibodies. In the ultimate affinity purification protocol, the desired antibody can be purified using a column of resin attached to the antigen against which the mAb was designed. For these last two methods, if the low pH is too harsh, a gentler high salt solution can be used to elute the antibodies from the column. Another tool in the antibody purification toolbox is precipitation. Antibodies precipitate out of solution at lower salt concentrations than many other proteins. Dialysis can remove the salt after precipitation. Electrophoresis is another tool for separating the antibody from contaminants by their sizes, or electrophoretic mobilities.
The biopharmaceutical industry has already optimized conditions for large-scale production of mAbs, and no doubt will continue to do so. Take advantage of the wealth of information that they have already provided us!
The image at the top of the page is from FiberCell Systems, Inc.