Purification of monoclonal antibodies

The same methods described for the purification of polyclonal anti- 

This method is less suitable for the purification of unprocessed 

A particular property of mammalian IgG is that their absorbance at 278 nm is about 2.5-3.0 higher than at 251 nm, in contrast to other serum proteins where this ratio is about 1-1.5 (in neutral buffers). Measurements at these two wavelengths may, therefore, indicate the fractions which contain IgG and their approximate degree of purity. The actual A2nl 42n ratio may vary for individual antibody clones. At the same time, the absorbance at 278 nm is an indication of the IgG concentration if the optical pathway is 1 cm, a 1% solution (10 mg/ml) has an absorbance of 13.5+ 10% (Little and Donahue, 1967). 

The SDS-PAGE method of Laemmli (1970) is convenient to verify the purity of the Ig, particularly with the very sensitive silver staining method (Section 16.1.1). It is good practice to analyse both unreduced and reduced (with 2-ME) samples. 

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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. 

Flatman S. Alam I. Gerard J. Mussa N. Process analytics for purification of monoclonal antibodies. Journal of Chromatography B, 2007, 848, 79-87. 

Tasaka, K Kobayashi, M Tanaka, T and Inagaki, C. (1984) Rapid purification of monoclonal antibody in ascites by high performance ion exchange column chromatography for diminishing non-specific staining. Acta Histochem. Cytochem. 17, 283-286. 

Figure 26-16 Purification of monoclonal antibody IgG by affinity chromatography on a 0.46 x 5 cm column containing protein A covalently attached to polymer support. Other proteins in the sample are eluted from 0 to 0.3 min at pH 7.6. When eluent pH is lowered to 2.6. IgG is freed from protein A and emerges from the column. [From B. J. Compton and L Kreilgaard. Chromatographic Analysis at Therapeutic Proteins," Anal. Chem. 1994,66, 1175A.]...

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... 

Note that so far the expanded bed mainly has been used with rather uncomplicated systems such as for purification of monoclonal antibodies from culture broth, isolation of extracellular substances from microbial cultivations, harvesting of fusion proteins from Escherichia coli cell homogenates, affinity isolation of certain enzymes from microbial homogenates, and separation of serum proteins from serum. To our knowledge there are very few reports on the isolation from homogenates of mammalian tissues or plant material. 

Levine, H. (1995). The use of membrane adsorbers for the purification of monoclonal antibodies. BioWest 95. 

Darby, C. R., Hamano, K., and Wood, K. J. (1993). Purification of monoclonal antibodies from tissue culture medium depleted of IgG.. Immunol. Methods 159, 125-129. 

Hakalahti, L., and Vihko, P. (1989). Purification of monoclonal antibodies raised against prostate-specific acid phosphatase for use in vivo in radioimaging of prostatic cancer. ]. Immunol. Methods 117, 131-136. 

Pavlu, B., Johansson, U., Nyhlen, C., and Wichman, A. (1986). Rapid purification of monoclonal antibodies by high-performance liquid chromatography. J. Chromatogr. 359, 449-460. 

Bukovsky, J., and Kennett, R. H. (1987). Simple and rapid purification of monoclonal antibodies from cell culture supernatants and ascites fluids by hydroxylapatite chromatography on analytical and preparative scales. Hybridoma 6, 219-228. 

Nopper, B., Kohen, F., and Wilchek, M. (1989). A thiophilic adsorbent for the one-step high-performance liquid chromatography purification of monoclonal antibodies. Anal. Biochem. 180, 66-71. 

Murray, A., Sekowski, M., Spencer, D. I., Denton, G., and Price, M. R. (1997). Purification of monoclonal antibodies by epitope and mimotope affinity chromatography. J. Chro-matogr A 782, 49-54. 

Burton, S. 2002. A generic approach to the purification of monoclonal antibodies An alternative to protein A. IBC Conference Antibody Production Downstream Processing, Feb 13-15, San Diego, CA. 

Purification of Monoclonal Antibody. Immunoglobulins were precipitated from the pooled ascites by addition of an equal volume of saturated ammonium sulfate [50% (NH4)2S04]. The precipitate was collected by centrifugation (20 min 10,240 X g), dissolved in 0.01 M sodium phosphate (pH 6.8), and reprecipitated. After the second ammonium sulfate precipitation, the pellet was dissolved in a minimum volume of 0.01 M sodium phosphate (pH 6.8) and centrifuged for 10 min at 10,600 X g). The resulting supemate was applied to a P6G, gel filtration polyacrylamid, column (Bio-gel Biorad, Rockville Center, NY 1.5 X 40 cm). Fractions containing protein were pooled and applied to a hydroxyapatite column that had been equilibrated with 0.01 M sodium phosphate (pH 6.8). Proteins were eluted with a linear gradient of 0.01 to 0.3 M sodium phosphate. 

CA Caulcott, R Boraston, C Hill, et al. Production and purification of monoclonal antibodies. In WP Collins, ed. Complementary Immunoassays. Chichester, UK Wiley, 1988, p 27. 

Chromatography steps for purification of monoclonal antibody produced by CHO cells Scale-down studies for resin use and viral clearance fully predictive of larger scale [26] (Genentech)... 

Francis R, Bonnerjea J, Hill C R (1990). Validation of the re-use of protein a sepharose for the purification of monoclonal antibodies. In D L Pyle (ed.). Separations for Biotechnology 2, Elsevier Applied Science, New York, pp. 491-498. 

Bukovsky, J., and Kennet, R. (1987). Simple and Rapid Purification of Monoclonal Antibodies from Cell Culture Supernatants and Ascites Fluids by Hydroxyl Apatite Chromatography on Analytical and Preparative Scales, Hybridoma 6 219-228. 

Yu DQ, McLean MD, HaU JC, Ghosh R. Purification of monoclonal antibody from tobacco extract using membrane-based bioseparation techniques. 7. Membr. Sci. 2008 323 159-166. 

Gottschlich, N. Kasche, V. Purification of monoclonal antibodies by simulated moving-bed chromatography.. Chromatogr. A, 1997, 765, 201-206. 

With affinity chromatography (AC), the biospecific interactions between, for example, a protein and the matching antibody are used to separate the protein or the antibody. This separation is based on a specific and reversible interaction between the molecules. A typical apphcation is the purification of monoclonal antibodies. Under the given conditions, they bond to a stationary phase that is covered with the protein A. After a washing step, the conditions are changed (often change in the pH value) such that the antibody elutes. 

Fig. 5 Purification of monoclonal antibody from cell culture supernatant by centrifugal precipitation chromatography.

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