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ABSTRACT
Exact expressions for the static light scattering of a solution containing up to three species of point-scattering solutes in highly nonideal solutions at arbitrary concentration are obtained from multicomponent scattering theory. Explicit expressions for thermodynamic interaction between solute molecules, required to evaluate the scattering relations, are obtained using an equivalent hard particle approximation similar to that employed earlier to interpret scattering of a single protein species at high concentration. The dependence of scattering intensity upon total protein concentration is calculated for mixtures of nonassociating proteins and for a single self-associating protein over a range of concentrations up to 200 g/l. An approximate semiempirical analysis of the concentration dependence of scattering intensity is proposed, according to which the contribution of thermodynamic interaction to scattering intensity is modeled as that of a single average hard spherical species. Simulated data containing pseudonoise comparable in magnitude to actual experimental uncertainty are modeled using relations obtained from the proposed semi-empirical analysis. It is shown that by using these relations one can extract from the data reasonably reliable information about underlying weak associations that are manifested only at very high total protein concentration.
INTRODUCTION
The quantitative characterization of proteins in highly concentrated solution is of interest for at least two reasons. one biological and one biotechnologies!. Some proteins, such as hemoglobin or crystallins. are present at concentrations of hundreds of grams/liter in their normal milieux, and small changes in their self-interaction resulting from changes in environmental variables or covalent modification may result in lhe formation of pathological aggregates ( 1 .2). When engineered antibodies are formulated as potential biopharmaceutical agents, attention must be paid to the effects of storage and administration in highly concentrated solution upon the association state of the protein and possible consequences for bioactivity and immunogenicity (3).
The characterization of weak associations leading to complex formation at high total protein concentration poses special problems to the experimenter. Weak attractive interactions are likely to be masked by large nonspecific repulsive interactions deriving from excluded volume. The formation of weakly associated complexes at high protein concentration, referred to as hidden associations (4), is revealed only when the contribution of repulsive, or nonideal. interactions to observed solution properties can be assessed explicitly. Techniques for detecting weak protein associations manifested...