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2 In Vitro Production of Monoclonal Antibody A major advantage of using mAb rather than polyclonal antiserum is the potential availability of almost infinite quantities of a specific monoclonal anti-body directed toward a single epitope (the part of an antigen molecule that is responsible for specific antigen-antibody interaction). In general, mAb are found either in the medium supporting the growth of a hybridoma in vitro or in ascitic fluid from a mouse inoculated with the hybridoma. mAb can be purified from either of the two sources but are often used as is in media or in ascitic fluid. In vitro methods should be used for final production of mAb when this is reasonable and practical. Many commercially available devices have been developed for in vitro cultivation. These devices vary in the facilities required for their operation, the amount of operator training required, the complexity of operating procedures, final concentration of antibody achieved, cost, and fluid volume accommodated. The cost of additional equipment should be considered in the cost of in vitro production methods. Each hybridoma cell line responds differently to a given in vitro production environment. This section describes in vitro production methods that are available and discusses the usefulness and limitations of each method. Batch Tissue-Culture Methods The simplest approach for producing mAb in vitro is to grow the hybridoma cultures in batches and purify the mAb from the culture medium. Fetal bovine serum is used in most tissue-culture media and contains bovine immunoglobulin at about 50μg/ml. The use of such serum in hybridoma culture medium can
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account for a substantial fraction of the immunoglobulins present in the culture fluids (Darby and others 1993). To avoid contamination with bovine immunoglobulin, several companies have developed serum-free media specifically formulated to, support the growth of hybridoma cell lines (Federspiel and others 1991; Tarleton and Beyer 1991; Velez and others 1986). In most cases, hybridomas growing in 10% fetal bovine serum (FBS) can be adapted within four passages (8–12 days) to grow in less than 1% FBS or in FBS-free media. However, this adaptation can take much longer and in 3–5% of the cases the hybridoma will never adapt to the low FBS media. After this adaptation, cell cultures are allowed to incubate in commonly used tissue-culture flasks under standard growth conditions for about 10 days; mAb is then harvested from the medium. The above approach yields mAb at concentrations that are typically below 20 μg/ml. Methods that increase the concentration of dissolved oxygen in the medium may increase cell viability and the density at which the cells grow and thus increase mAb concentration (Boraston and others 1984; Miller and others 1987). Some of those methods use spinner flasks and roller bottles that keep the culture medium in constant circulation and thus permit nutrients and gases to distribute more evenly in large volumes of cell-culture medium (Reuveny and others 1986; Tarleton and Beyer 1991). The gas-permeable bag (available through Baxter and Diagnostic Chemicals), a fairly recent development, increases concentrations of dissolved gas by allowing gases to pass through the wall of the culture container. All these methods can increase productivity substantially, but antibody concentrations remain in the range of a few micrograms per milliliter (Heidel 1997; Peterson and Peavey 1998; Vachula and others 1995). Most research applications require mAb concentration of 0.1–10 mg/ml, much higher than mAb concentrations in batch tissue-culture media (Coligan and others). If unpurified antibodies are sufficient for the research application, low-molecular-weight cutoff filtration devices that rely on centrifugation or gas pressure can be used to increase mAb concentration. Alternatively, tissue-culture supernatants can be purified by passage over a protein A or protein G affinity column, and mAb can then be eluted from the column at concentrations suitable for most applications (Akerstrom and others 1985; Peterson and Peavey 1998). However, bovine or other immunoglobulin present in the culture medium will contaminate the monoclonal antibody preparation. Either concentration step can be performed in a day or less with minimal hands-on time. In short, batch tissue-culture methods are technically relatively easy to perform, have relatively low startup costs, have a start-to-finish time (about 3 weeks) that is similar to that of the ascites method, and make it possible to produce quantities of mAb comparable with those produced by the mouse ascites method. The disadvantages of these methods are that large volumes of tissue-culture media must be processed, the mAb concentration achieved will be low (around a few micrograms per milliliter), and some mAb are denatured during concentration or purification (Lullau and others 1996). In fact, a random screen of mAb
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revealed that activity was decreased in 42% by one or another of the standard concentration or purification processes (Underwood and Bean 1985). Semipermeable-Membrane-Based Systems As mentioned above, growth of hybridoma cells to higher densities in culture results in larger amounts of mAb that can be harvested from the media. The use of a barrier, either a hollow fiber or a membrane, with a low-molecular-weight cutoff (10,00–G30,000 kD), has been implemented in several devices to permit cells to grow at high densities (Evans and Miller 1988; Falkenberg and others 1995; Jackson and others 1996). These devices are called semipermeable-membrane-based systems. The objective of these systems is to isolate the cells and mAb produced in a small chamber separated by a barrier from a larger compartment that contains the culture media. Culture can be supplemented with numerous factors that help optimize growth of the hybridoma (Jaspert and others 1995). Nutrient and cell waste products readily diffuse across the barrier and are at equilibrium with a large volume, but cells and mAb are retained in a smaller volume (0–5 ml in a typical membrane system or small hollow-fiber cartridge). Expended medium in the larger reservoir can be replaced without losing cells or mAb; similarly, cells and mAb can be harvested independently of the growth medium. This compartmentalization makes it possible to achieve mAb concentrations comparable with those in mouse ascites. Two membrane-based systems are available: the mini-PERM® (Unisyn Technologies, Hopkinton, MA) and the CELLine® (Integra Bioscience, Ijamsville, MD). The CELLine has the appearance of and is handled similarly to a standard T Flask but is separated into two chambers by a semi-permeable membrane and a gas-permeable membrane is on its underside next to the cell chamber. The mini-PERM has a similar design but is cylindrical and comes with a motor unit that functions to roll the fermentor continuously to allow gas and nutrient distribution. Startup for these units costs about $300–800 and requires a C02 incubator. The advantage of membrane-based systems is that high concentrations of mAb can be produced in relatively low volumes and fetal calf serum can be present in the media reservoir with only insignificant crossover of bovine immunoglobulins into the cell chamber. A disadvantage is that the mAb may be contaminated with dead cell products. Technical difficulty is slightly more than that of the batch tissue-culture methods but should not present a problem for laboratories that are already doing tissue culture. The total mAb yield from a membrane system ranges from 10–160 mg according to Unisyn literature. In the hollow-fiber bioreactor, medium is continuously pumped through a circuit that consists of a hollow-fiber cartridge, gas-permeable tubing that oxygenates the media, and a medium reservoir. The hollow-fiber cartridge is composed of multiple fibers that run through a chamber that contains hybridoma cells growing at high density. These fibers are semipermeable and serve a purpose
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similar to that of membrane-based systems. The hollow-fiber bioreactor is technically the most difficult of in vitro systems, partly because of the susceptibility of cells grown at extremely high density to environmental changes and toxic metabolic-byproduct buildup. The hollow-fiber bioreactor is designed to provide total yields of 500 mg mAb or more. Startup of this kind of system usually costs more than $1,200. For those reasons, hollow-fiber reactors are used only if large quantities of mAb are needed. The hollow-fiber reactor has been successfully used in many independent laboratories (Jackson and others 1996; Knazek and others 1972; Peterson and Peavey 1998). If investigators are unable to invest the time or material costs, several institutional core facilities and government and commercial contract laboratories produce mAb from a hybridoma. For example, commercial contract laboratories typically charge $1 1/mg to produce 1,000 mg with hollow-fiber reactors (Chandler, 1998). Recently, several workshops, forums, and publications have discussed the use of the alternative methods to replace mice for production of mAb (Center for Alternatives to Animal Testing and OPRR/NIH 1997; Marx and others 1997; de Geus and Hendriksen, eds 1998). Their conclusions indicate that alternative methods can often provide an adequate means of generating most of the mAb needed by the research community. In vitro methods for producing mAb are appropriate in numerous situations, and it is the responsibility of the researcher to produce scientific justification for using the mouse ascites method. It is the responsibility of the IACUC to evaluate researchers' scientific justification and to approve or disapprove the use of mouse ascites methods.
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