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Phase Behavior of an Intact Monoclonal Antibody

Tangir Ahamed ^* , Beatriz N. A. Esteban ^* , Marcel Ottens ^*, Gijs W. K. van Dedem ^*, Luuk A. M. van der Wielen ^*, Marc A. T. Bisschops , Albert Lee , Christine Pham  and Jörg Thömmes 

^* Department of Biotechnology, Delft University of Technology, Delft, The Netherlands; Biogen Idec, San Diego, California; and Xendo, Leiden, The Netherlands

Correspondence: Address reprint requests to Marcel Ottens, Dept. of Biotechnology, Delft University of Technology, Delft, The Netherlands. E-mail: m.ottens{at}tudelft.nl.

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 (B₂₂) 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. B₂₂ 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 B₂₂ 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 B₂₂ units also supports the existence of a universal phase diagram of a complex glycoprotein when it is recast in a protein interaction parameter.