Characterization of Opalescence in low Volume Monoclonal Antibody Solutions Enabled by Microscale Nephelometry.

Monoclonal antibody (mAb)-based drugs are often prone to unfavorable solution behaviors including high viscosity, opalescence, phase separation, and aggregation at the high concentrations needed to enable patient-centric subcutaneous dosage forms. Given that these can have a detrimental impact on manufacturability, stability, and delivery, approaches to identifying, monitoring, and controlling these behaviors during drug development are critical. Opalescence presents a significant challenge due to its relationship to liquid-liquid phase separation. Quantitative characterization of opalescence via turbidimetry is often restrictive due to large volume requirements (>2 mL) and alternative microscale approaches based on light transmittance (Eckhardt et al., J Pharm Sci Technol. 1994, 48: 64-70) may pose challenging with respect to accuracy. To address the need for accurate and quantitative microscale opalescence measurements, we have evaluated the use of a 'de-tuned' static light scattering detector which requires <10 µL sample per measurement. We show that tuning of the laser power to a range far below that of traditional light scattering measurements results in a stable detector response that can be accurately calibrated to the nephelometric turbidity unit (NTU) scale using appropriate standards. The calibrated detector signal yields NTU values for mAbs and other protein solutions that are comparable to a commercial turbidimeter. We used this microscale approach to characterize the opalescence of 48 commercial mAb drug products and found that the majority have opalescence below 15 NTU. However, in products with mAb concentrations greater than 75 mg/mL, a broad range of opalescence was observed, in a few cases greater than 20 NTU. These measurements as well as nephelometric characterization of several IgG1 and IgG4 mAbs across a broad pH range highlight subclass-specific tendencies toward opalescence in high concentration solutions.

A single molecular descriptor to predict solution behavior of therapeutic antibodies.

Despite the therapeutic success of monoclonal antibodies (mAbs), early identification of developable mAb drug candidates with optimal manufacturability, stability, and delivery attributes remains elusive. Poor solution behavior, which manifests as high solution viscosity or opalescence, profoundly affects the developability of mAb drugs. Using a diverse dataset of 59 mAbs, including 43 approved products, and an array of molecular descriptors spanning colloidal, conformational, charge-based, hydrodynamic, and hydrophobic properties, we show that poor solution behavior is prevalent (>30%) in mAbs and is singularly predicted (>90%) by the diffusion interaction parameter (k D), a dilute-solution measure of colloidal self-interaction. No other descriptor, individually or in combination, was found to be as effective as k D. We also show that well-behaved mAbs, a substantial subset of which bear high positive charge and pI, present no disadvantages with respect to pharmacokinetics in humans. Here, we provide a systematic framework with quantitative thresholds for selecting well-behaved therapeutic mAbs during drug discovery.