Abstract

The Huggins coefficient kH 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 kH measurements have been reported for select monoclonal antibodies (mAbs) solutions, there has been limited study of kH 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 kH 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 kH 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 kH from the standpoint of fundamental protein–protein interactions. Despite shortcomings in our theoretical understanding of kH 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.

Highlights

  • The antibodies were expressed by Chinese Hamster Ovary (CHO) cells, purified by protein A chromatography followed by a polishing step consisting of ion exchange resin chromatography, and concentrated in their respective formulation buffer to 100 mg/mL nominal protein concentration (Table 1)

  • Theratio ratio monomer and aggregate content is wellinappreciated in dedetermining the shear viscosity of protein and antibody solutions, which necessitates the termining the shear viscosity of protein and antibody solutions, which necessitates the need to make Size Exclusion Chromatography (SEC) measurements [8,16,93,94]

  • Kastelic et al find kH to equal 0.6 near the theta point where Ψ vanishes, which is in the range (0.5, 0.57) of the estimates of kH for the polymer model. This deviation is understandable given the lack of consideration of charge, dipole interactions, hydration, and other factors known to contribute to protein–protein interactions in protein solutions

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Summary

Introduction

Recombinant proteins such as immunoglobulins (IgGs) are routinely administered to patients at relatively high protein concentrations, often exceeding 100 mg/mL. Protein therapeutics provide relatively high in vivo potency, due to their large molecular weights and high required doses on the order of. Required for subcutaneous drug administration [6] At these high protein concentrations, the protein drug may “degrade” through aggregation and high solution viscosity, which are common challenges that must be overcome [1,7,8]. The aggregation of a protein biotherapeutic can lead to reduced drug product potency and a greatly increased immunogenic response upon administration to patients [9,10,11]. The development of metrologies for quantifying protein–protein interactions, and for anticipating changes in the viscosity of protein solutions with changes in protein concentration and other changes in processing and clinical conditions, is a topic of intense scientific research and practical technological interest

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