Monoclonal chromatography

Immunoaffinity chromatography utilizes the high specificity of antigen—antibody interactions to achieve a separation. The procedure typically involves the binding, to a soHd phase, of a mouse monoclonal antibody which reacts either directly with the protein to be purified or with a closely associated protein which itself binds the product protein. The former approach has been appHed in the preparation of Factor VIII (43) and Factor IX (61) concentrates. The latter method has been used in the preparation of Factor VIII (42) by immobilization of a monoclonal antibody to von WiHebrand factor [109319-16-6] (62), a protein to which Factor VIII binds noncovalenfly. Further purification is necessary downstream of the immunoaffinity step to remove... 

Hydrophobic interaction chromatography (HIC) can be considered to be a variant of reversed phase chromatography, in which the polarity of the mobile phase is modulated by adjusting the concentration of a salt such as ammonium sulfate. The analyte, which is initially adsorbed to a hydrophobic phase, desorbs as the ionic strength is decreased. One application demonstrating extraordinary selectivity was the separation of isoforms of a monoclonal antibody differing only in the inclusion of a particular aspartic acid residue in the normal, cyclic, or iso forms.27 The uses and limitations of hydrophobic interaction chromatography in process-scale purifications are discussed in Chapter 3. 

Gretch, D.R., Suter, M., and Stinski, M.F. (1987) The use of biotinylated monoclonal antibodies and streptavidin affinity chromatography to isolate herpesvirus hydrophobic proteins or glycoproteins. Anal. Biochem. 163, 270-277. 

Liquid chromatography-electrogenerated chemiluminescence Lithium diisopropylamide Low-density lipoprotein Laser-induced fluorescence Limit of detection Monoclonal antibody Maximum admissible concentration l,2-B (3-chlorophenyl)ethylenediamine Micellar electrokinetic chromatography 4,4 - Oxalyl 6 [(trifluoromethylsulfonyl)imino]-ethylene 6 (4-methylmorpholinium)trifluoromethanesulfonate Maltose phosphorylase Multipinned phase... 

Donohue-Rolfe, Arthur, David W.K. Acheson, Anne V. Kane, and Gerald T. Keusch. "Purification of Shiga Toxin and Shiga-Like Toxins I and II by Receptor Analog Affinity Chromatography with Immobilized PI Glycoprotein and Production of Cross-Reactive Monoclonal Antibodies." Infection and Immunity 57 (December 1989) 3888-893. 

Factor IX may also be purified by immunoaffinity chromatography, using immobilized anti-IX murine monoclonals. Purification to homogeneity is particularly important in the case of... 

Immunoaffinity chromatography (IAC), 6 400—402 12 137, 145 Immunoanalyzers, automated, 14 150 Immunoassay(s), 14 135-159. See also Immunoassay- DNA probe hybrid assays Immunoassay methods Immuno(bio)sensors antibody-antigen reaction, 14 136-138 basic technology in, 14 138-140 chemiluminescent, 14 150-151 classification of, 14 140-153 design of, 14 139-140 enzyme, 14 143-148 fluorescence, 14 148-150 highly specific, 14 153 historical perspective on, 14 136 microarrays and, 14 156—157 microfluidics in, 26 968—969 monoclonal versus polyclonal antibodies in, 14 152-153... 

Fig. 9. Conjoint Liquid Chromatography (CLC). Separation of proteins from mouse ascites and isolation of monoclonal antibody IgG in one step obtained by a combination of CIM QA and CIM Protein A Disks. Conditions Separation mode CLC (first disk CIM QA, 12 x 3 mm ID, 0.34 ml second disk - CIM Protein A, 12 x 3 mm ID, 0.34 ml, inserted in monolithic column housing) Instrumentation Gradient HPLC system with extra low dead volume mixing chamber Sample Mouse ascites Injection volume 20 pL Mobile Phase Buffer A 20 mM Tris-HCl, pH 7.4 Buffer B Buffer A + 1 M NaCl Buffer C 0.1 M Acetic acid Conditions Gradient 0 - 50 % B in 50 s, 100% A for 40 s, 100% C for 30 s Flow Rate 4 ml/min Detection UV at 280 nm... 

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. 

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

A., and Roepstorff P. (2001), Characterization of a recombinant monoclonal antibody by mass spectrometry combined with bquid chromatography. J. Chromatogr. 

CZE and cIEF are also used as complementary tools with traditional ion-exchange chromatography to monitor the charge heterogeneity of monoclonal antibodies. Advantages... 

A third area of development in carbohydrate l.c. analyses is in the combined techniques (see Section IV,3) and other methods that provide qualitative, as well as quantitative, information about sample constituents, such as high-performance liquid affinity chromatography. The use of specific lectin- and monoclonal antibody-based, stationary phases for analytical and preparative applications is now being considered. The basic concepts of these techniques have been reviewed - and their applications to carbohydrates have been discussed. 

Burchiel S. W Billman, J. R and Alber, T. R. (1984) Rapid and efficient purification of mouse monoclonal antibodies from ascites fluid using high performance liquid chromatography. J. Immunol. Methods 69, 33-42. 

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. 

---