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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