AF4 and PEG Precipitation as Predictive Assays for Antibody Self-Association.

Monoclonal antibodies (mAbs) are often formulated as high-protein-concentration solutions, which in some cases can exhibit physical stability issues such as high viscosity and opalescence. To ensure that mAb-based drugs can meet their manufacturing, stability, and delivery requirements, it is advantageous to screen for and select mAbs during discovery that are not prone to such behaviors. It has been recently shown that both these macroscopic properties can be predicted to a certain extent from the diffusion interaction parameter (kD), which is a measure of self-association under dilute conditions.1 However, kD can be challenging to measure at the early stage of discovery, where a relatively large amount of a high-purity material, which is required by traditional methods, is often not available. In this study, we demonstrate asymmetric field-flow fractionation (AF4) as a tool to measure self-association and therefore identify antibodies with problematic issues at high concentrations. The principle lies on the ability to concentrate the sample close to the membrane during the injection mode, which can reach formulation-relevant concentrations (>100 mg/mL).2 By analyzing a well-characterized library of commercial antibodies, we show that the measured retention time of the antibodies allows us to pinpoint molecules that exhibit issues at high concentrations. Remarkably, our AF4 assay requires very little (30 µg) sample under dilute conditions and does not need extensive sample purification. Furthermore, we show that a polyethylene glycol (PEG) precipitation assay provides results consistent with AF4 and moreover can further differentiate molecules with issues of opalescence or high viscosity. Overall, our results delineate a two-step strategy for the identification of problematic variants at high concentrations, with AF4 for early developability screening, followed by a PEG assay to validate the problematic molecules and further discriminate between opalescence or high-viscosity issues. This two-step antibody selection strategy enables us to select antibodies early in the discovery process, which are compatible with high-concentration formulations.

Cytotoxicity of doxorubicin-conjugated poly[N-(2-hydroxypropyl)methacrylamide]-modified γ-Fe2O3 nanoparticles towards human tumor cells.

Doxorubicin-conjugated magnetic nanoparticles containing hydrolyzable hydrazone bonds were developed using a non-toxic poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) coating, which ensured good colloidal stability in aqueous media and limited internalization by the cells, however, enabled adhesion to the cell surface. While the neat PHPMA-coated particles proved to be non-toxic, doxorubicin-conjugated particles exhibited enhanced cytotoxicity in both drug-sensitive and drug-resistant tumor cells compared to free doxorubicin. The newly developed doxorubicin-conjugated PHPMA-coated magnetic particles seem to be a promising magnetically targeted vehicle for anticancer drug delivery.