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Up to now, the productivity of mammalian cell culture has been perceived as limiting the productivity of the industrial manufacture of therapeutic monoclonal antibodies. Dramatic improvements in cell culture performance have changed this picture, and the throughput of antibody purification processes is gaining increasing attention. Although chromatographic separations currently are the centerpiece of antibody purification, mostly due to their high resolving power, it becomes more and more apparent that there may be limitations at the very large scale. This review will discuss a number of alternatives to chromatographic antibody purification, with a particular emphasis on the ability to increase throughput and overcome traditional drawbacks of column chromatography. Specifically, precipitation, membrane chromatography, high-resolution ultrafiltration, crystallization, and high-pressure refolding will be evaluated as potential large scale unit operations for industrial antibody production.

Understanding protein phase behavior is important for purification, storage, and stable formulation of protein drugs in the biopharmaceutical industry. Glycoproteins, such as monoclonal antibodies (MAbs) are the most abundant biopharmaceuticals and probably the most difficult to crystallize among water-soluble proteins. This study explores the possibility of correlating osmotic second virial coefficient (B(22)) with the phase behavior of an intact MAb, which has so far proved impossible to crystallize. The phase diagram of the MAb is presented as a function of the concentration of different classes of precipitants, i.e., NaCl, (NH4)2SO4, and polyethylene glycol. All these precipitants show a similar behavior of decreasing solubility with increasing precipitant concentration. B(22) values were also measured as a function of the concentration of the different precipitants by self-interaction chromatography and correlated with the phase diagrams. Correlating phase diagrams with B(22) data provides useful information not only for a fundamental understanding of the phase behavior of MAbs, but also for understanding the reason why certain proteins are extremely difficult to crystallize. The scaling of the phase diagram in B(22) units also supports the existence of a universal phase diagram of a complex glycoprotein when it is recast in a protein interaction parameter.

Membrane chromatography was introduced as an integrative technology for the purification of proteins several years ago. The main feature of chromatographic separations based on membranes is the absence of pore diffusion, which is the main transport resistance in conventional column chromatography using porous particles. This is achieved by attaching the active ligands to the inner surface of the through‐pores of microfiltration membranes, where mass transport takes place mainly by convective flow, thus reducing the transport limitations from pore to film diffusion. In combination with a low pressure drop across a membrane, very high volumetric flows are possible in a membrane‐based purification step; thus, the time requirement of a complete chromatographic cycle is reduced. The method is characterized by fast processing at preserved or even increased resolution compared to standard chromatography on particulate materials. Since its introduction, many successful applications of membrane chromatography have been described. Additionally, theoretical analysis has been performed, which has significantly helped to understand present and visualize future applications of this technology. In this review, the fundamental theoretical considerations from the actual literature are described, and then the applications of membrane‐based separation processes are presented. We have attempted to demonstrate the usefulness of this new technology in the purification of many interesting proteins.

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