Feasibility of polyelectrolyte-driven Fab fragment separation

The use of antigen-binding fragments (Fabs) as biotherapeutic agents is gaining interest and thus requires development of adequate purification strategies aimed at separating Fabs from other proteins. Thus, the feasibility of using a copolymer for separation of Fabs from monoclonal antibodies (mAbs) and fragment constant regions (Fcs) was evaluated, employing a blend of purified solutions of these proteins. The use of a copolymer exerting both hydrophobic as well as anionic properties resulted in high precipitation yields for both the mAb and Fc fragment, even at ionic strength of 150 mM NaCl. On the contrary, Fabs exhibited reduced precipitation yields upon copolymer addition. These observations are attributed to differences in protein physicochemical parameters, allowing mAbs and Fcs to be precipitated via conjoint electrostatic and hydrophobic interactions. In contrast, Fabs were mainly precipitated via electrostatic interactions, being reduced at higher ionic strength. This finding was corroborated by hydrophobicity analysis using 2-p-toluidinonaphthalene-6-sulfonate, showing enhanced hydrophobicity of Fcs compared to mAbs alone, while Fabs exhibited the lowest hydrophobicity. Within the context of increasing demand for Fabs as therapeutic proteins, these results may open up a simpler purification strategy for this protein class, potentially also to be implemented within the context of polymer-driven protein purification during fermentation.