Monoclonal antibodies cell purification

Clausen, A. M., Subramanian, A., and Carr, P. W., Purification of monoclonal antibodies from cell culture supernatants using a modified zirconia-based cation-exchange support, /. Chromatogr. A, 831, 63, 1999. 

Proteins are frequently powerful immunogens and the availability of specific antibodies, particularly monoclonal antibodies, makes the technique of affinity chromatography very useful in the separation and purification of individual proteins. The technique has been used to purify a wide range of proteins such as hormones, membrane receptors and complement proteins. However, it is not restricted to proteins and is potentially applicable to any immunogenic substance. The availability of suitable antibodies is essential and these may be raised by whole animal polyclonal techniques or by monoclonal cell culture. The former antibodies may need some prior purification before being immobilized. 

Purification of murine antiheparin monoclonal antibody produced in cell culture was monitored by Malsch et al.63 using a CZE method with a borate or boric acid buffer (pH 9) in an uncoated capillary. 

On the other hand, certain tumor cells have the traits for immortality and rapid proliferation. By combining the two cells by fusion, a hybridoma can be created that has both traits. The monoclonal antibodies (MAbs) produced from the hybridoma cells can be used for diagnosis, disease treatment, and protein purification. 

The most commonly used technique to produce bispecific antibodies from two monoclonal antibodies is by fusing two hybridoma cell lines by conventional cell fusion procedure (Staerz and Bevan, 1986). These cells produce all possible combinations of the heavy and light chains of both antibodies, including the desired bispecific antibody. A limitation is that only part of the antibodies is the desired bispecific monoclonal antibody therefore, further purification is necessary (Van Ravenswaay et al., 1993). 

Thommes J, Born C, Biselli M, Wandrey C, Kula MR (1995) Purification of monoclonal antibodies by fluidized bed adsorption. In Beuvery CE, Griffiths JB, Zeijlmaker WP (ed) Animal cell technology developments towards the 21st century. Kluwer, p 515... 

Affinity chromatography techniques have shown less utility in analytical testing than in preparative separations for a variety of reasons, including cost and the difficulty of validating consistent operation as the column changes over time. Protein A affinity has been commonly used to quantitate the total antibody content of either ascites or cell culture fluids. To provide guidance in the development of a purification process, specific immunoaffinity resins are either available or can be readily prepared to quantitate the levels of unrelated protein contaminants. To rapidly determine what the active species in a mixture is, a monoclonal antibody that... 

Steroid hormones achieve their effects on target tissues through intracellular receptor proteins. According to recent views, oestrogen and progestin receptors are localized in the nuclear compartment of the cells, whereas glucocorticoid receptors may reside in both the cytoplasm and the nucleus. Determination of the intracellular localization of androgen receptors awaits the development of (monoclonal) antibodies which will enable immunohistochemical studies. The molecular aspects of the mechanism of action of steroid hormones will be covered in other chapters [1-3] in this volume. The present chapter deals with the characterization, assay and purification of steroid receptors. 

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