KJ-N and TV-J were supported by a NOVA grant from Aarhus University Research Foundation
KJ-N and TV-J were supported by a NOVA grant from Aarhus University Research Foundation. Funding Statement This work was supported by the National Institutes of Health [EB000092-02]. Author contributions SKC, KJ-M, AN, HZ, and PS designed and carried out experiments; AP, SD-O, RE provided materials. to deliver injectable therapeutic doses is a key requirement for the development of stable, safe, and efficacious antibody therapeutics. Interactions that control macromolecular solution behavior span a large affinity range and the resulting solution structures can span an exceptionally large size range. Far-field interactions that modulate the molecular distance distribution in solution, as well as short-range weak interactions that lead to transient complexes, may cause phase separation, promote the formation of immunogenic irreversible aggregates, or cause excessive solution viscosity.1C8 In the search for conditions of pH, ionic strength, and excipients that yield safe and efficacious formulations, powerful biophysical methods have been used by different laboratories to predict and characterize higher-order structures and interactions of protein pharmaceuticals, including computational approaches,5,9-11 and experimental techniques such as static and dynamic light scattering, small-angle scattering, analytical ultracentrifugation, and chromatography.12C21 However, a key experimental difficulty for the characterization of weak protein interactions with any technique is the need to study concentrated solutions that are thermodynamically and hydrodynamically nonideal. For this discussion, we adopt a platform accounting for reversible self-association explicitly as oligomeric claims linked by mass action regulation, and independent from nonideality, which is here understood as causes that modulate interparticle range distribution without leading to physical complex formation, such as volume exclusion and long-range repulsive or attractive relationships.22 For fundamental reasons, this nonideality prohibits standard polydispersity analysis due to the hydrodynamic coupling of all macromolecular motion, and the concomitant violation of the linear superposition basic principle.23 Although, in basic principle, nonideal solution behavior can be modeled, e.g., in scattering and sedimentation techniques, such models require assumptions within the existence Dynasore of one or a few discrete species, and the potential effect of polydispersity within the measurement remains uncertain. Consequently, the inability to account simultaneously for polydispersity and protein interactions in nonideal macromolecular solutions offers substantially hampered the study of restorative formulations in the high concentrations within the order of 100 mg/mL typically required. For dilute antibody solutions, typically up to a few mg/mL, sedimentation velocity analytical ultracentrifugation (SV) is definitely a gold standard for quantitation of trace aggregates, orthogonal to size-exclusion chromatography (SEC).24,25 To briefly recapitulate the physical basis, separation is accomplished in SV by virtue of the high centrifugal field during ultracentrifugation that causes strongly size-dependent migration of macromolecular particles free in solution, and prospects to the formation of sedimentation boundaries. These are optically measured and mathematically modeled to determine the diffusion-deconvoluted sedimentation coefficient distributions and of 4.0 (blue); apparent sedimentation coefficient distribution at 1.6 as measured in dilute remedy by ideal and of 3 mL/g, suggest slightly attractive interactions. Open in a separate window Number 2. Assessment of measured sedimentation boundaries of mAb A (a) and mAb D (b). Concentrations are 37 mg/mL for mAb A and 46 mg/mL for mAb D, and sedimentation at 45,000 rpm is definitely displayed by every 10th data point (points) of every 3rd scan (time intervals of 120?sec). The solid collection is the best-fit nonideal and (observe Table 1). This results in a percentage of rmsd/loading transmission of 0.42% for mAb A and 0.41% for mAb D. The related best-fit sedimentation coefficient distributions (symbols) and best-fit self-association models (lines) for different mAbs. and as they do about (e.g., to a small value of log10(and optimizing both and and resulting from the 4.8((could not be determined and was Dynasore fixed to HSP90AA1 upper limit. (d) In the isotherm analysis, the portion of incompetent irreversible dimer could not be independently identified and was fixed to the value determined from and as a function of loading concentration can be interpreted Dynasore like a reflection of mass action law shifting the time-average human population of different claims. These isotherms of for the equilibrium dissociation constant and lower case for the nonideality coefficients of diffusion); they adhere to the empirical rating from comparison of the.