Wei-Jie Fang

Insulin is an indispensable cornerstone in the management of diabetes mellitus, yet its extreme susceptibility to aggregation under mechanical stress remains a formidable challenge for long-term formulation stability. This study investigates the potential of Poloxamer 188 (P188) as a chemically stable alternative to the industry-standard Polysorbate 20 (PS20) for stabilizing insulin. Formulations containing graded concentrations of P188 (0.005%−0.15%, w/v), 0.05% (w/v) PS20, or no surfactant were subjected to 24 h shaking stress at 300 rpm. Our results reveal a striking concentration-dependent profile for P188, where 0.05% PS20 consistently maintains robust protection, whereas low-to-moderate concentrations of P188 (0.005%−0.05%) fail to stabilize insulin and instead exert a pronounced “pro-aggregatory” effect. At 0.05% P188, this pro-aggregatory behavior leads to severe macroscopic turbidity, a massive surge in particle concentration (≥2 μm), and the near-total disappearance of detectable insulin monomers in the supernatant. Crucially, elevating the P188 concentration to 0.15% effectively prevents macroscopic turbidity and preserves high soluble protein recovery. Mechanistically, the instability observed at low-to-moderate P188 concentrations stems from its sluggish interfacial adsorption kinetics compared to PS20, leaving nascent air-liquid interfaces temporarily unprotected. Under such conditions, P188 functions as a macromolecular scaffold and nucleation core, cross-linking interface-denatured proteins with native molecules to drive rapid co-assembly. These findings serve as a critical warning for the biopharmaceutical industry that the use of P188 in insulin-based products must be approached with extreme caution to avoid inadvertent protein aggregation.

Higher-order antibody aggregates remain challenging to characterize using conventional mass spectrometry because of their low abundance, broad mass distributions, and extensive heterogeneity. Here, the practical performance of a commercial electrostatic linear ion trap charge detection mass spectrometer (Xevo ^TM CDMS) was evaluated for therapeutic antibody analysis. The platform provided reproducible mass and charge measurements, with repeatability and intermediate-precision RSDs below 1%, and maintained consistent mass determination across the concentration and solution conditions examined. Controlled mixtures of antibody-derived species showed a linear molar-ratio-dependent ion response, supporting semi-quantitative estimation of relative species abundance under defined conditions. For heat-stressed trastuzumab, CDMS measurements were qualitatively consistent with SEC-MALS, mass photometry, and conventional native MS, while enabling direct mass determination of individual ions within highly heterogeneous high-molecular-weight (HMW) populations that exhibited extensive charge-state overlap and could not be reliably deconvoluted by conventional native MS. High-duty-cycle acquisition increased sampling of low-abundance, high- m/z ions and improved access to higher-order aggregate signals. Joint single-ion mass–charge analysis further differentiated statistical ion families within the broad HMW fraction and related major mass differences to independently obtained compositional information. These results support commercial Xevo ^TM CDMS as a practical multidimensional platform for antibody aggregate characterization.

High-concentration protein formulations for subcutaneous administration have developed rapidly, while viscosity-related pipetting errors remain a key bottl

Driven by the evolving demands of modern pharmaceutical industry, high-concentration monoclonal antibody formulations (HCMFs) have emerged as a central research hotspot in biopharmaceutical development, attributable to their merits such as low administration volume and reduced medical expenditure. For lyophilized HCMFs, reconstitution performance serves as a critical quality attribute of equivalent importance to long-term storage stability. As components in direct contact with lyophilized drug products, primary packaging containers are indispensable critical excipients for pharmaceutical preparation. This work systematically investigated the effects of inner surface characteristics of glass vials on the long-term stability and reconstitution behaviors of lyophilized HCMFs. The results demonstrated that the surface hydrophilicity/hydrophobicity of vial walls exerted no remarkable influence on the physicochemical stability of mAb-X during prolonged storage. Nevertheless, in the reconstitution process, the intrinsically high viscosity of concentrated mAb solutions, combined with the ultralow surface free energy, intense hydrophobicity and weakened contact line pinning effect of such vials, triggered massive visible-bubbles to adhere beneath the liquid–solid interface. These trapped bubbles failed to ascend and dissipate along the inner walls and bottom of vials. Collectively, this study verifies that hydrophobic primary packaging materials, when adopted as storage containers for lyophilized HCMFs, will induce severe reconstitution defects, which may further lead to inaccurate dosing and compromised clinical operability.

Messenger RNA–lipid nanoparticles (mRNA-LNPs) are commonly stored frozen and distributed via cold-chain logistics, yet how freezing conditions affect their post-thaw quality attributes remains insufficiently understood. In this study, two liquid mRNA-LNP formulations, without sucrose or containing 10% sucrose, were subjected to repeated freeze–thaw cycles under three representative freezer conditions (−10, −30, and −80 °C). Particle size, zeta potential, entrapment efficiency, mRNA integrity, and functional activity were then assessed. Different freezing conditions produced divergent post-thaw outcomes. Rapid freezing better preserved particle size, mRNA integrity, and functional activity, but was accompanied by lower encapsulation retention and greater payload leakage. Conversely, slower or intermediate freezing better preserved encapsulation in some cases, yet more readily compromised mRNA integrity and activity. By integrating electron paramagnetic resonance, differential scanning calorimetry, and time–temperature profiling, we propose that these divergent outcomes arise from two partially decoupled damage pathways. Rapid freezing may induce stronger acute osmotic/interfacial perturbation during solidification, thereby impairing membrane barrier function and reducing encapsulation retention. However, rapid freezing also shortened residence in the mobile freeze-concentrated phase, limiting cumulative microenvironmental stress and better preserving mRNA integrity and functional activity. In contrast, slower or intermediate freezing prolonged residence in this phase, promoting molecular damage and functional loss. These findings suggest that although transient osmotic/interfacial shock may contribute to encapsulation loss during rapid freezing, final translational activity is more closely determined by stress accumulation during residence in the mobile freeze-concentrated phase, providing a mechanistic basis for optimizing freezing conditions for liquid mRNA-LNP formulations.