Mechanistic Investigation of Zn(II) Extraction and Interactions in Combination Biological Drug Products

Prefilled syringes (PFS) and autoinjectors are increasingly used to deliver biologic drug products, particularly monoclonal antibodies (mAbs), to improve convenience, compliance, and dosing accuracy. However, these systems face performance and quality challenges such as needle clogging, particularly for high-concentration, viscous, and aggregation-prone formulations. The origins and mechanisms by which device-derived leachables influence clogging and protein stability remain poorly understood. In this study, we investigated zinc (Zn) leaching from rigid needle shields (RNS) and its interactions with a high-concentration dupilumab formulation and common excipients. Inductively coupled plasma-mass spectrometry (ICP-MS) quantification shows that Zn was the predominant metal in RNS batches, and its extraction kinetics depended strongly on time, temperature, and the presence of a routinely utilized surfactant, polysorbate 80 (PS-80). Stressing RNS materials in the dupilumab formulation at 40 °C for 14 days yielded up to 550 μg/mL Zn(II), roughly 100-fold above typical specifications. Isothermal titration calorimetry (ITC) revealed millimolar Zn(II) binding to PS-80 and weaker interaction with buffer components (histidine, arginine, and acetate), which together promote Zn release. Structural modeling identified surface-exposed regions of dupilumab enriched in histidine-, sulfur-, and carboxylic-acid-containing residues that are geometrically arranged to chelate Zn(II), highlighting likely Zn(II) binding motifs. These protein-metal and excipient-metal interactions, along with PS-80 degradation catalyzed by Zn, correlate with increased solution viscosity and the formation of high-molecular-weight species. Zn leaching from RNS and its synergistic interactions with PS-80, buffer components, and the mAb can drive PS-80 degradation, increase viscosity, and promote higher-order protein aggregation, factors that plausibly contribute to needle clogging. Overall, Zn(II) can simultaneously interact with proteins, increasing their propensity to aggregate while degrading the excipients intended to stabilize them against aggregation. Understanding these mechanisms can inform candidate selection, formulation design, and device choice to mitigate protein aggregation and syringe clogging and improve product reliability and therapeutic outcomes.