Biopharmaceutical Evaluation of Intermolecular Interactions by AUC-SE

Abstract

Analytical ultracentrifugation sedimentation equilibrium (AUC-SE) is a useful technique to investigate the weak reversible intermolecular interactions among proteins in solution. It provides biophysical information such as the average apparent molecular weight, stoichiometry, and association constant of associating proteins. Several studies of intermolecular interaction in biopharmaceuticals by AUC-SE are introduced in this chapter. AUC-SE also provides the second virial coefficient (B 2 ), which represents the type, i.e., repulsive and attractive, and a magnitude of intermolecular interactions. The B 2 values obtained from the protein solution at low concentrations showed good correlation with aggregation and viscosity of MAb at high concentrations, indicating that B 2 can be an effective indicator of aggregation propensity and viscosity. These findings suggest that AUC-SE provides clues to understand the self-association in biopharmaceuticals and to establish effective manufacturing process, formulation, and administration of biopharmaceuticals.

Mitigation of liquid–liquid phase separation of a monoclonal antibody by mutations of negative charges on the Fab surface

“…A k D value of −20 mL/g was a threshold for LLPS where the B 2 value indicated zero in this case study. Previous studies [ 28 – 30 , 35 – 38 ] have also demonstrated a linear relationship between the k D and B 2 values. The investigations by Connolly et al [ 28 ] indicated this relationship among eight different mAbs and corroborated our results.…”

“…The B 2 was −4.08 × 10 −5 mL·mol/g 2 and the k D was −33 mL/g for the WT. The sign for B 2 suggests the existence of attractive protein–protein intermolecular interactions [ 11 , 20 , 21 , 26 , 29 , 30 ]. The addition of 150 mM NaCl increased the k D value from −33 mL/g to −12 mL/g, indicating that the protein–protein self–attractive interactions in low–ionic–strength buffer A could essentially be mediated by electrostatic interactions.…”

“…The B 2 values for these mAbs were determined using the AUC-SE method [ 28 – 30 ] ( Table 1 ). A negative B 2 value denotes attractive protein–protein interactions, whereas a positive value denotes mutual repulsion.…”

“…We determined apparent molecular weights by nonlinear least-squares fitting using Origin software (OriginLab Corporation, Northampton, MA, USA). B 2 was obtained from the slope of the plot between the inverse of the apparent molecular weight and protein concentration, as shown by Saito et al [ 29 , 30 ]. Because of limited sample availability, the experiment was performed in one replicate.…”

Sedimentation velocity analytical ultracentrifugation for characterization of therapeutic antibodies

“…Composition gradient static light scattering (CG-SLS) is another approach to investigate self-and hetero-associations of proteins at high concentration (Attri and Minton 2005). By using CG-SLS, the second virial coefficient of the IgG-IgG interaction was determined over the range of concentrations up to >150 mg/mL (Kress et al 2016), allowing assessment of the extent of intermolecular interactions and thus being a useful tool for the rational formulation development (Saito and Uchiyama 2016).…”