The currently prevalent matrices for chromatographic separation of immunoglobulins (Igs) are based on Protein A or its recombinant versions (Protein G). They display excellent selectivity and specificity, but are expensive. A Protein A matrix costs $8,000 to 12,000 per L-resin. The typical production column volume is 100 L. The million-dollar matrix is far more expensive than the production hardware.
Research work with human and humanized antibodies and the development of novel types of hybrid monoclonals will require the development of separation methods not based on protein A/G, because protein A/G does not recognize IgG from all species. Although Protein A chromatography is the leading choice for antibody purification, it is not the unanimous choice. The most frequently mentioned alternatives are ion exchange, hydrophobic interaction chromatography, and even relatively crude methods like ammonium sulfate and caprylic acid precipitation. Purification schemes for antibodies from the serum include precipitation,7,8,9 ion-exchange chromatography,10,11 thiophilic chromatography,12,13 metal chelate interaction chromatography,14,15 affinity separations using immobilized Protein-A/G,1,16 hydrophobic interaction chromatography,17,18 hydroxyapatite chromatography,19,20 dye affinity, and ion-exchange techniques.21–24
PROPERTIES OF DESIRABLE SUPPORTS Economics, efficiency, and practicality are some of the constraints centered around the search for novel chromatographic supports and methodologies. The preparation of alternative stationary phase supports is an important area. The goal of our research is to develop support materials that offer novel selectivities, or develop new protocols that are amenable to scaleup without presenting excessive operational complexities. We focused on developing adsorbents that have a narrow range of physiochemical affinities and on mixed-mode synthetic chemistries coupled with engineered matrices.
In this article, we at the University of Nebraska explore and examine recent progress in the development of pseudoaffinity methods and the synthesis of engineered matrices. We discuss its use in the purification of antibodies from biological sources. In the first case study, the development of ligand-modified chitosan as a stationary phase material will be an example of a methodology based on pseudoaffinity and as so can be utilized to separate and purify MAbs from cell culture supernatant. In the second case study, the development of zirconia as a stationary-phase material is an example where engineered matrices in combination with mixed-mode chemistries can be utilized to separate humanized MAbs from cell culture supernatant. The named products were purchased for use in an academic setting.