Characterization and stabilization of recombinant human protein pentraxin (rhPTX-2).

The potential human therapeutic protein pentraxin (PTX-2) is a large, glycosylated plasma protein consisting of five monomers that self-associate noncovalently into a pentameric, ring-like structure. The structural integrity and conformational stability of recombinant human PTX-2 (rhPTX-2) as a function of temperature and pH is described in conjunction with the identification of stabilizing excipients that improve the protein's physical stability. The monomer consisted primarily of two glycosylated forms (25,171 and 25,462 Da) as determined by mass spectrometry. The protein was approximately 82%-97% pentamer in solution with smaller amounts of decamer and aggregate as measured by analytical ultracentrifugation and size-exclusion chromatography (SEC). Comprehensive biophysical characterization using an empirical phase diagram approach demonstrates that rhPTX-2 is conformationally stable over a wide pH range (6.0-8.5) and at temperatures below 65°C-75°C. Formation of soluble aggregates was identified as a major physical degradation pathway of rhPTX-2 in solution under accelerated storage conditions. A series of effective stabilizers was identified by SEC analysis including disaccharides, sugar alcohols, citrate, as well as neutral and charged amino acids. The preformulation characterization data and stability profile presented in this work enable future studies for rhPTX-2 formulation development.