Monoclonal antibody interactions and phase behavior

Protein phase behavior is involved in numerous aspects of downstream processing, either by design as in crystallization or precipitation processes, or as an undesired effect, such as aggregation. An improved understanding of protein phase behavior is therefore important for developing rational design strategies for important process steps. This work explores the phase behavior of four monoclonal antibodies (mAbs) that exhibit liquid-liquid separation, aggregation, gelation, and crystallization. A systematic study of a number of factors, including solution composition and pH, has been conducted in order to explore the phase behavior of these antibodies.;IDEC-152 is discussed first and used as a basis of comparison for the behavior of the other antibodies. The phenomena observed include a significant dependence of the cloud point on the cation in sulfate salts, and a non-monotonic pH dependence. Additionally, the first crystallization of this mAb is reported. Phase separation of Mab A was found under a much wider range of solution conditions than observed for IDEC-152. The behavior of Mab B showed that this antibody behaved similarly to IDEC-152 with respect to phase boundary locations, but a significantly different dense phase than observed for any other antibody was identified. IDEC-151 is not discussed independently as the small quantities available limited its study; however, it is included in the discussion comparing the antibodies studied, for the conditions for which data were available.;Comparisons of the four antibodies from the perspective of phase boundary locations show consistent trends as a function of solution composition; however, changing solution pH has different effects on each of the antibodies studied. Further, the dense phases formed varied among the antibodies. Protein-protein interactions, as indicated by values of the osmotic second virial coefficient, are used in an attempt to correlate the phase behavior. The primary finding is that values of the osmotic second virial coefficient are useful for correlating phase boundary locations, but do not provide a strong correlation with the type of dense phase that results.;Monoclonal antibody oligomers were studied as an additional method to understand phase behavior, specifically the types of dense phases formed. Oligomers and aggregates are known to play a large role in pharmaceutical processing and have been implicated in some physiological and pathological conditions. The underlying causes and effects of this behavior remain poorly understood. Using dynamic light scattering, the four monoclonal antibodies were found to form reversible oligomers in concentrated lithium sulfate solutions at neutral pH. This behavior depended on the protein concentration, the salt type and concentration, and on the solution pH. The oligomers were characterized by analytical ultracentrifugation and found to consist primarily of dimers and trimers. Three of the four antibodies studied behaved similarly, while the fourth formed only dimers in a wider range of solution conditions. In most cases oligomerization appears to inhibit protein crystallization, though the dimers formed by the fourth antibody did not.