Chromatographic Purification of MAbs with Non-Affinity Supports

Purifying MAbs with novel supports works as well as Protein A.
Jun 01, 2005
Volume 18, Issue 6

Anu Subramanian, Ph.D.
The introduction of new protein-based therapeutics such as monoclonal antibodies (MAbs), MAb-based vaccines, growth factors and plasma proteins implies the need to study, characterize, and purify. The separation step is likely to be a bottleneck and cost-effective technology will be needed to rectify it.

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.

While affinity chromatography using Protein A/G is specific, both Protein A and Protein G are macromolecular and fragile, expensive to obtain from bacterial or tissue-culture sources, and are difficult to immobilize without losing activity. The conditions used to elute IgG or an antibody bound to protein-A and protein-G sorbents are harsh and require a pH near 3.0. This can impact the biological activity of some antibody products. Hence, the product is immediately neutralized and dialyzed against a pH 7.0 buffer. To overcome this negative effect, recombinant versions of protein-A and protein-G have been developed that elute at milder conditions in the 6.0 to 7.0 pH range.

In addition, the use of both proteins as affinity supports in chromatographic columns poses special challenges regarding regeneration and sanitation.1 Some of these drawbacks preclude the use of biological ligands in practical applications and has prompted many researchers to turn their attention to the development of synthetic ligands. Smaller molecules like dyes, amino acids, metal ions, and chemical moieties show comparable affinities, and specificity of such a molecule can be increased or decreased, either at adsorption or desorption, to attain resolutions and degrees of purification comparable to those of immunoadsorption.2–6

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.

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