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Comparison of Huggins Coefficients and Osmotic Second Virial Coefficients of Buffered Solutions of Monoclonal Antibodies.

ABSTRACT: The Huggins coefficient k_H is a well-known metric for quantifying the increase in solution viscosity arising from intermolecular interactions in relatively dilute macromolecular solutions, and there has been much interest in this solution property in connection with developing improved antibody therapeutics. While numerous k_H measurements have been reported for select monoclonal antibodies (mAbs) solutions, there has been limited study of k_H in terms of the fundamental molecular interactions that determine this property. In this paper, we compare measurements of the osmotic second virial coefficient B₂₂, a common metric of intermolecular and interparticle interaction strength, to measurements of k_H for model antibody solutions. This comparison is motivated by the seminal work of Russel for hard sphere particles having a short-range "sticky" interparticle interaction, and we also compare our data with known results for uncharged flexible polymers having variable excluded volume interactions because proteins are polypeptide chains. Our observations indicate that neither the adhesive hard sphere model, a common colloidal model of globular proteins, nor the familiar uncharged flexible polymer model, an excellent model of intrinsically disordered proteins, describes the dependence of k_H of these antibodies on B₂₂. Clearly, an improved understanding of protein and ion solvation by water as well as dipole-dipole and charge-dipole effects is required to understand the significance of k_H from the standpoint of fundamental protein-protein interactions. Despite shortcomings in our theoretical understanding of k_H for antibody solutions, this quantity provides a useful practical measure of the strength of interprotein interactions at elevated protein concentrations that is of direct significance for the development of antibody formulations that minimize the solution viscosity.

SUBMITTER: Pathak JA

PROVIDER: S-EPMC7922252 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Antibody-based therapeutics are the fastest-growing drug class on the market, used to treat aggressive forms of cancer, chronic autoimmune conditions, and numerous other disease states. Although the specificity, affinity, and versatility of therapeutic antibodies can provide an advantage over traditional small-molecule drugs, their development and optimization can be much more challenging and time-consuming. This is, in part, because the ideal formulation buffer systems used for in vitro characterization inadequately reflect the crowded biological environments (serum, endosomal lumen, etc.) that these drugs experience once administered to a patient. Such environments can perturb the binding of antibodies to their antigens and receptors, as well as homo- and hetero-aggregation, thereby altering therapeutic effect and disposition in ways that are incompletely understood. Although excluded volume effects are classically thought to favor binding, weak interactions with co-solutes in crowded conditions can inhibit binding. The second virial coefficient (B2) parameter quantifies such weak interactions and can be determined by a variety of techniques in dilute solution, but analogous methods in complex biological fluids are not well established. Here, we demonstrate that fluorescence correlation spectroscopy is able to measure diffusive B2-values directly in undiluted serum. Apparent second virial coefficient (B2,app) measurements of antibodies in serum reveal that changes in the balance between attractive and repulsive interactions can dramatically impact global nonideality. Furthermore, our findings suggest that the approach of isolating specific components and completing independent cross-term virial coefficient measurements may not be an effective approach to characterizing nonideality in serum. The approach presented here could enrich our understanding of the effects of biological environments on proteins in general and advance the development of therapeutic antibodies and other protein-based therapeutics.

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Comparison of Huggins Coefficients and Osmotic Second Virial Coefficients of Buffered Solutions of Monoclonal Antibodies.

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