Abstract
The study of weak or colloidal interactions of therapeutic proteins in different formulations allows prediction and optimization of protein stability. Various biophysical techniques have been applied to determine the second osmotic virial coefficient B2 as it reflects on the macromolecular distance distribution that governs solution behavior at high concentration. In the present work, we exploit a direct link predicted by hydrodynamic theory between B2 and the nonideality of sedimentation, commonly measured in sedimentation velocity analytical ultracentrifugation through the nonideality coefficient of sedimentation, kS. Using sedimentation equilibrium analytical ultracentrifugation for independent measurement of B2, we have examined the dependence of kS on B2 for model proteins in different buffers. The data exhibit the expected linear relationship and highlight the impact of protein shape on the magnitude of the nonideality coefficient kS. Recently, measurements of kS have been considerably simplified allowing higher throughput and simultaneous polydispersity assessment at higher protein concentrations. Thus, sedimentation velocity may offer a useful approach to compare the impact of formulation conditions on weak interactions and simultaneously on higher-order structure of therapeutic proteins.
Highlights
Protein pharmaceuticals are a rapidly expanding class of therapeutics
B2 = 0 we can derive an experimental estimate of the nonideality of sedimentation for vanishing virial coefficients, k0S (Eq 7), which exclusively reports on the shape dependence of the hydrodynamic interactions
These values are all close to the value of 9.1 ml/g predicted for the hydrodynamically equivalent sphere of antibodies
Summary
Protein pharmaceuticals are a rapidly expanding class of therapeutics. In particular, monoclonal antibody products have found broad application with an increasing number of targets [1,2,3]. One of the challenges is the development of formulations that provide colloidal stability, low solution viscosity, and ensure the absence of immunogenic aggregates at very high protein concentrations [4,5,6,7]. Techniques that can assess both the formation of higher-order structure and measure weak particle interactions are of great interest in this field. It has been shown that the second virial coefficient B2 can correlate with aggregation, viscosity, solubility, and liquid-liquid phase separation [8,9,10,11,12,13,14,15]. Techniques used to measure B2 include small angle scattering, static light scattering (SLS), sedimentation equilibrium (SE) and sedimentation velocity (SV) analytical ultracentrifugation (AUC), self-interaction chromatography, osmometry, and dynamic light scattering (DLS) in combination with SV [9,13,15,16,17,18,19,20,21,22,23,24,25,26]
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