Hydrophobic interaction chromatography antibodies

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. 

Antibodies are proteins well adapted for hydrophobic interaction chromatography because they have specific conserved zones with high density of hydrophobic aminoacids (see Fig. 1). It has been shown that IgG have 18 highly conserved hydrophobic sites, most of them located close to Cy2 and Cy3 domains, and are rich in valine, leucine, isoleucine, and alanine.110,111 These sites are also contributing to the interaction of antibodies with protein A from S. aureus (see Section V.F). 

Manzke, O., Tesch, H., Diehl, V., and Bohlen, H. (1997). Single-step purification of bispecific monoclonal antibodies for immunotherapeutic use by hydrophobic interaction chromatography. J. Immunol. Methods 208, 65-73. 

Figure 10 Separation of a monoclonal antibody by hydrophobic interaction chromatography. IgG is eluted during the decreasing gradi at a salt concentration of about 500 mM ammonium sulfate.

Hydrophobic interaction chromatography (HIC) is a powerful method for resolving all types of biomolecules. Immimoglobulins have strongly hydro-phobic domains compared with the majority of typical contaminants and so this technique is especially appropriate. My impression is that it is not routinely used by many investigators or companies who supply antibodies for the re-... 

Azevedo AM, Rosa PAJ, Ferreira LF, Aires-Barros MR. Integrated process for the purification of antibodies combining aqueous two-phase extraction, hydrophobic interaction chromatography and size-exclusion chromatography. J Chromatogr A 2008 1213(2) 154-61. 

Chapter 15 provides an in-depth coverage of various chromatographic methods, such as ion exchange, hydrophobic interaction, affinity, ligand, immunoaffinity, gel filtration, etc., that can be used for separations of antibodies by liquid chromatography. 

A hydrophobic interaction membrane chromatography method for rapid and efficient separation and analysis of monoclonal antibodies aggregates, able to resolve Campath-IH monomer, dimer, trimer, tetramer, and pentamer, was recently reported [269]. Other applications of hydrophobic interaction membrane chromatography include the separation of model proteins [270], the purification of humanized monoclonal antibody using a stack of microporous synthetic membranes [271], and the fractionation of human plasma proteins using a 0.1 pm microporous PVDF membrane [272]. 

Ghosh R, Wang L. Purification of humanized monoclonal antibody by hydrophobic interaction membrane chromatography. 7. Chromatogr. A 2006 1107 104-109. 

IMAC is particularly suitable for the separation of biomolecules such as antibodies because it can be performed under very mild, nondenaturing conditions. Single-step purification is often possible for antibodies produced in cell culture supernatants. However, monoclonal antibodies produced in ascites fluid often need an additional step to remove nonspecific IgG and other contaminants. A combination of IMAC with hydrophobic interaction or ion-exchange chromatography would provide an excellent method for antibody purification [71]. 

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