| dc.description.abstract | Understanding and overcoming analytic, formulation, manufacturing, and regulatory challenges of protein drugs or biosimilars covers the latest trends and challenges of both academic and R&D of pharmaceutical companies. The focus are on: understanding and controlling protein aggregation, improving detection and quantitation of aggregates, analyzing subvisible and visible particles with various techniques, understanding aggregates as an inducing factor in immungenicity, and improving structural analysis and modeling to predict protein aggregation. In a word to describe all the above attentive focus is “second virial coefficient B22” of protein in solution.
Here we cope with the theoretical developments of the current methods for B22 measurements, especially focus is on Self-interaction Chromatography (SIC). Moreover, based on our extensive experience on Isothermal Titration Calorimetry (ITC), we derive a statistic thermodynamics model to obtain B22 of protein to describe protein behaviors in solution by means of the dilution enthalpy measurement of protein solution. And, the B22 value can be applied for the protein purifcation, protein conformational disease (PCD) and protein crystallization.
In this work, we focus on the studies of solution additive effects on B22, including NaCl, n-propanol, and Arginine. It shows that protein-protein interactions change from repulsive(electrostatic dominant) to attractive(hydrophobic dominant) with increasing NaCl conc. from the ITC experimental result. From the study of organic solvent effect, Protein-protein repulsive interactions increased with increasing np. conc. at the lower conc. range; however, protein-protein attractive interactions increased with increasing np. conc. was shown at the range of higher np. conc. Amino acid effect on B22 of protein in solution exhibits that protein-protein interactions aren’t function of Arg. conc. as the present of lower NaCl conc.(2% w/v). But, protein-protein repulsive interactions increased with increasing Arg. conc. at the solution condition of NaCl conc.5% (w/v).
Comparison ITC experimental results with SIC, we can discover that unexpected results occur at SIC system as the surface charge distribution of protein isn’t uniform and the pH of solution is closer to the pI of protein due to immobilization of protein molecules on the solid support. The orientation concern of the immobilized protein molecules is the most drawback for SIC methodology to determine B22. On the contrary, we can describe precisely protein behaviors in solution by ITC because of the fully degree of rational freedom of protein at ITC solution system. Besides, the B22 values measured by ITC has the advantage on costs including the materials and time, especially for the studies of those pharmaceutical proteins.
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