Glycation and oxidation: key determinants of the physicochemical behaviour of trastuzumab deruxtecan (Enhertu^®) in a glucose infusion solution

Introduction
Trastuzumab deruxtecan (T-DXd) is an anti-HER2 antibody-drug conjugate (ADC) for which stability following dilution remains poorly documented, despite being of major interest to hospital pharmacy departments responsible for preparing the drug. The aim of this study was to evaluate the physicochemical and functional stability of T-DXd diluted in 5% glucose (G5%) under conditions representative of clinical practice.

Materials and methods
Three batches of T-DXd at concentrations of 1 and 4 mg/mL were prepared in the presence of G5%, in multilayer polypropylene bags, and then stored at 2–8 °C or at 25 °C for 28 days. An orthogonal analytical strategy was implemented to monitor the key critical quality attributes: pH, visual appearance, turbidity, colloidal stability (DLS), aggregation and fragmentation (SEC), hydrophobicity (HIC), glycosylation profile (HILIC), charge heterogeneity (CEX), structural modifications (LC-MS) and biological activity (HER2 ELISA and FcR affinity). The integrity of the conjugate and the payload-linker system was assessed.

Results
The pH remained stable (5.4–5.5) and no visible particles or increase in turbidity were observed. The levels of intact antibodies, as measured by Protein A affinity chromatography, remained stable (< 5 % variation) with no significant effect of concentration, temperature or batch. SEC, HIC, DLS and HILIC analyses revealed no aggregation, fragmentation or changes to the glycosylation profile. The integrity of the conjugate and the linker-payload system was preserved, with no signs of deconjugation or loss of payload. In contrast, CEX analysis revealed a gradual increase in acidic variants at 25 °C from Day 21 onwards. LC-MS analyses confirmed the onset of oxidation and glycation from Day 14 at room temperature, whilst no significant changes were observed when stored under refrigeration. These alterations were accompanied by a reduction in HER2-binding activity at 25 °C (78% residual activity on day 28 compared with 94% at 2–8 °C).

Conclusion
T-DXd diluted in G5% retains its physicochemical, structural and functional characteristics for at least 28 days at 2–8 °C. At room temperature, gradual chemical changes affect biological activity after one week of storage. These results support the feasibility of prolonged storage of diluted T-DXd preparations in hospital pharmacies under refrigerated conditions.