Understanding and controlling aggregation is an essential aspect in the development of pharmaceutical proteins to improve product yield, potency and quality consistency. Even a minute quantity of aggregates may be reactogenic and can render the final product unusable. Self-assembly processing of virus-like particles (VLPs) is an efficient method to quicken the delivery of safe and efficacious vaccines to the market at low cost. VLP production, as with the manufacture of many biotherapeutics, is susceptible to aggregation, which may be minimized through the use of accurate and practical mathematical models. However, existing models for virus assembly are idealized, and do not predict the non-native aggregation behavior of self-assembling viral subunits in a tractable nor useful way. Here we present a mechanistic mathematical model describing VLP self-assembly that accounts for partitioning of reactive subunits between the correct and aggregation pathways. Our results show that unproductive aggregation causes up to 38% product loss by competing favorably with the productive nucleation of self-assembling subunits, therefore limiting the availability of nuclei for subsequent capsid growth. The protein subunit aggregation reaction exhibits an apparent second-order concentration dependence, suggesting a dimerization-controlled agglomeration pathway. Despite the plethora of possible assembly intermediates and aggregation pathways, protein aggregation behavior may be predicted by a relatively simple yet realistic model. More importantly, we have shown that our bioengineering model is amenable to different reactor formats, thus opening the way to rational scale-up strategies for products that comprise biomolecular assemblies.

A gradient of negative surface charge based on 1-D spatial variation from surface sulfhydryl to mixed sulfhydryl-sulfonate moities was prepared by controlled UV oxidation of 3-mercaptopropylsilane monolayer on fused silica. Adsorption of three human plasma proteins, albumin (HSA), immunoglobulin G (IgG), and fibrinogen (Fgn) onto such surface gradient was studied using spatially-resolved total internal reflection fluorescence (TIRF) and autoradiography. Adsorption was measured from dilute solutions equivalent to 1/100 (TIRF, autoradiography), and 1/500 and 1/1000 (autoradiography) of protein's physiological concentrations in plasma. All three proteins adsorbed more to the non-oxidized sulfhydryl region than to the oxidized, mixed sulfhydryl-sulfonate region of the gradient. In the case of HSA the adsorption contrast along the gradient was largest when the adsorption took place from more dilute protein solutions. Increasing the concentration to 1/100 of protein plasma concentration eliminated the effect of the gradient on HSA adsorption and to the lesser extent on IgG adsorption. In the case of Fgn the greatest adsorption contrast was observed at the highest concentration used. Based on adsorption kinetics, the estimated binding affinity of HSA for the sulfhydryl region what twice the affinity for the mixed sulfhydryl-sulfonate region of the gradient. For IgG and Fgn the initial adsorption was transport-limited and the initial adsorption rates approached the computed flux of the protein to the surface.

Competitive adsorption of three human plasma proteins: albumin (HSA), fibrinogen (Fgn), and immunoglobulin G (IgG) from their ternary solution mixtures onto a sulfhydryl-to-sulfonate gradient surface was investigated using spatially-resolved total internal reflection fluorescence (TIRF) and autoradiography. The concentration of each protein in the ternary solution mixture was kept at an equivalent of 1/100 of its physiological concentration in blood plasma. The three proteins displayed different adsorption and desorption characteristics. Each protein adsorbed less to the sulfonate region than to the sulfhydryl region of the gradient. The adsorption-desorption kinetics revealed large differences in the adsorption and desorption rates of three proteins. By fitting the experimental data to a simple model of competitive protein adsorption, the affinity of each protein to the surface at the gradient center position was ranked as: Fgn > HSA ≫ IgG. Competitive exchange of adsorbed proteins was related to the magnitude of desorption rate constants. Such competitive adsorption of the three major human plasma proteins illustrates the complex dynamics of blood proteins – biomaterials interactions.

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