High-throughput analysis of concentration-dependent antibody self-association.

Monoclonal antibodies are typically monomeric and nonviscous at low concentrations, yet they display highly variable associative and viscous behavior at elevated concentrations. Although measurements of antibody self-association are critical for understanding this complex behavior, traditional biophysical methods are not capable of characterizing such concentration-dependent self-association in a high-throughput manner. Here we describe a nanoparticle-based method, termed self-interaction nanoparticle spectroscopy, that is capable of rapidly measuring concentration-dependent self-interactions for three human monoclonal antibodies with unique solution behaviors.

Surface interactions of monoclonal antibodies characterized by quartz crystal microbalance with dissipation: impact of hydrophobicity and protein self-interactions.

Surface adsorption of two monoclonal antibodies (mAb1 and mAb2), with widely different hydrophobicity and self-association behavior in solution, was examined by quartz crystal microbalance with dissipation monitoring to understand how adsorption and protein self-interactions near the surface are impacted by their intrinsic properties. The dependence of mass and viscoelastic properties of the adsorbed protein layer on the type of surface, presence of a surfactant, protein concentration, and pH were examined. Adsorption was significantly reduced in the presence of surfactant for both proteins, but for the more hydrophobic mAb2, residual adsorption remained on polystyrene (PS) and Teflon surfaces.

Quantification and characterization of subvisible proteinaceous particles in opalescent mAb formulations using micro-flow imaging.

Micro-flow imaging (MFI) has been shown to be more sensitive than light obscuration (LO) methods for measuring subvisible proteinaceous particles in protein formulations. Given the potential challenges in detecting particulates in opalescent mAb formulations, the accuracy of MFI to size and count particles in opalescent solutions was investigated and compared to LO and membrane microscopy methods. Proteinaceous monoclonal antibody (mAb) particles, generated either by chemical denaturation or agitation stress, polystyrene and glass particles were used as model systems for measurements in opalescent mAb solutions.

Quantitation of aggregates in therapeutic proteins using sedimentation velocity analytical ultracentrifugation: practical considerations that affect precision and accuracy.

Aggregation is a major degradation pathway that needs to be characterized and controlled during the development of protein pharmaceuticals. Analytical ultracentrifugation-sedimentation velocity (AUC-SV) is emerging as an important orthogonal tool to size exclusion chromatography to quantitate aggregates. However, the precision and accuracy of modern AUC-SV and the experimental variables that influence these two performance parameters need to be better understood and controlled.

Phage passage after extended processing in small-virus-retentive filters.

Retention of a two small phages (PhiX-174 and pp7) by direct-flow small-virus-retentive filters [Viresolve NFP (normal-flow parvovirus), Virosart CPV (canine parvovirus), Ultipor DV20 and Planova 20N] was studied using a commercial-process fluid. Phage passage occurred in each filter type, particularly when overloaded with phage. Clearances of pp7 and PhiX-174 were similar for any given filter brand, arguing that the two phages are equivalent for testing small-virus-retentive filters.

Non-native intermediate conformational states of human growth hormone in the presence of organic solvents.

Purpose: Manufacturing processes expose protein pharmaceuticals to organic solvents that may perturb the native folded state, increasing the potential for irreversible aggregation or surface adsorption. The aim of this study was to characterize the conformational states of human growth hormone (hGH) in aqueous ethanolic solutions.

Methods: The higher order structure of hGH was investigated using far- and near-UV circular dichroism (CD) and fluorescence spectroscopy as orthogonal techniques, and the hydrodynamic size was monitored using dynamic light scattering.

Opalescent appearance of an IgG1 antibody at high concentrations and its relationship to noncovalent association.

Purpose: Therapeutic antibodies are often formulated at a high concentration where they may have an opalescent appearance. The aim of this study is to understand the origin of this opalescence, especially its relationship to noncovalent association and physical stability.

Methods: The turbidity and the association state of an IgG1 antibody were investigated as a function of concentration and temperature using static and dynamic light scattering, nephelometric turbidity, and analytical ultracentrifugation.

Hybrid insulin cocrystals for controlled release delivery.

The ability to tailor the release profile of a drug by manipulating its formulation matrix offers important therapeutic advantages. We show here that human insulin can be cocrystallized at preselected ratios with the fully active lipophilically modified insulin derivative octanoyl-N(epsilon)-LysB29-human insulin (C8-HI). The cocrystal is analogous to the NPH (neutral protamine Hagedorn) crystalline complex formed with human insulin, which is commonly used as the long-acting insulin component of diabetes therapy.