Abstract This study investigates sugar-mediated modulation of intermolecular positional correlations in monoclonal antibody (mAb) formulations upon freeze-concentration using small-angle X-ray scattering (SAXS). While previous studies have focused on solutions or dried states, experimental data on freeze-concentrated matrices (FCMs) for mAbs are still scarce, representing a key knowledge gap. Understanding the mAb FCM is key for the formulation of frozen drug substance, frozen drug product, and lyophilizates. To address this gap, we combined complementary systems: a solution concentration series of constant composition but varying total solid content, ice-free reconstituted FCM analogues, and genuine in situ frozen FCMs with varying sucrose/mAb ratios, measured within the ice-containing, non-equilibrium state. The concentration series reproduces the compositional upconcentration of all solutes that occurs upon freezing and enables tracking how protein−protein correlation features evolve with increasing crowding. A controlled cooling ramp from 20 to −40 °C probed the effect of temperature on intermolecular organization in FCM analogues and their response to cooling across Tg′. Structure factor features indicated a progressive transition from whole-molecule spacing to shorter-range, domain-level correlations with increasing concentration, converging on the distances observed in the FCM analogues and, as confirmed by cross-comparison, in the genuine in situ frozen FCM. A complementary, geometry-based coarse-grained 12-bead modelserving as an interpretive geometric frameworkreproduced the experimental correlation lengths, supporting their interpretation as sub-molecular proximity between neighboring subunits or domains. In the FCM analogues and in situ frozen FCMs, the sucrose-to-mAb ratio was a primary factor modulating intermolecular spacing, while buffer conditions had no measurable impact. Together, the FCM analogues and in situ frozen measurements provide mechanistic insight into protein packing in freeze-concentrated environments relevant for formulation stability.
Abstract 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.
Abstract Wet granulation is an interfacial reconstruction process mediated by liquid binders. However, the mechanisms underlying the distinct granulation behaviors of drugs with different solubilities remain poorly understood due to a lack of molecular-level insight. Here, we elucidate these mechanisms using density functional theory (DFT) calculations and molecular dynamics (MD) simulations. Three model drugs with distinct solubilitiesaminophylline (AMI), metformin hydrochloride (MET), and diclofenac sodium (SD)were selected. The results show that differences in granulation behavior cannot be attributed solely to solubility; rather, they arise from variations in interfacial interactions may be governed by crystal structure. Molecular simulations further reveal that crystal structure contributes to macroscopic granulation outcomes by regulating the dynamics of interfacial water molecules.
Abstract Coformulated monoclonal antibodies (mAbs) offer a promising next-generation therapeutic strategy by combining multiple antibodies in a single drug product to enhance efficacy and improve patient compliance. However, ensuring the quality and stability of individual components in combination remains a key challenge. Here, we systematically characterized ten IgG1 mAbs (eight low-viscosity and two high-viscosity mAbs at 150 mg/mL) and all 45 possible 1:1 binary coformulations. Viscosity measurements revealed three distinct cross-interaction patterns: (1) minimal cross-interaction, where coformulation viscosity fell between those of the parental mAbs; (2) attractive cross-interaction, characterized by viscosity above both parental mAbs; and (3) repulsive cross-interaction, characterized by viscosity below both parental mAbs. Among the two high-viscosity mAbs, one exhibited reduced viscosity when coformulated with low-viscosity mAbs, whereas the other exhibited increased viscosity. Diffusion interaction parameter (kD) measurements showed partial correspondence with the observed interaction patterns, although kD did not consistently align with the viscosity trends across all coformulation pairs. In parallel, conformational stability was assessed using thermal unfolding parameters, including onset temperature (Tonset) and the first unfolding transition temperature (Tm1). Most coformulations exhibited thermal unfolding parameters intermediate between those of their parental mAbs, often shifting toward the less conformationally stable parent, indicating that conformational stability was generally retained following coformulation. This study represents one of the most comprehensive investigations of antibody coformulations to date and, to our knowledge, is the first to report repulsive interactions, particularly in the context of high-concentration viscosity. These findings highlight fundamental protein−protein interaction patterns that govern antibody coformulations, expand the knowledge base for the development of stable next-generation antibody combination therapies, and provide a rich experimental dataset to guide future computational modeling of mAb−mAb coformulation behavior.
Abstract Affinity hydrogels enable precise control of cargo release kinetics through specific, non-covalent interactions between the functionalized polymer network and therapeutic agents. Yet, how these interactions integrate with steric hindrance in the crosslinked network to determine cargo diffusivity, a critical kinetic parameter governing cargo-controlled release, remains poorly quantified. Here, we present a theoretical framework that explicitly bridges microscopic cargo−polymer interactions with macroscopic cargo release kinetics. Specifically, our approach delineates cargo diffusivity into two principal contributions: (i) binding interactions and (ii) steric hindrance, enabling a quantitative understanding of their interplay in defining cargo release performance. Increasing binding interactions, attractively, reduce diffusivity in a reverse sigmoidal fashion, transitioning from a steric- to a binding-controlled regime. Overall, we demonstrate how the interplay of microscopic forces governs cargo diffusivity within affinity hydrogels and, in turn, regulates cumulative cargo release at the macroscopic level, thereby bridging microscopic thermodynamic interactions with macroscopic cargo release kinetics, quantitatively.
