ObjectiveThe primary objective of this experimental formulation-development study was to prepare and physicochemically characterize a trastuzumab-loaded liposomal formulation containing post-inserted DSPE-PEG2000 for systemic-delivery research. The secondary objective was to determine whether the preparation and recovery process preserved trastuzumab monomeric integrity and HER2-binding activity.MethodsLiposomes were prepared by thin-film hydration, pulsed probe sonication, sequential membrane extrusion, post-insertion of DSPE-PEG2000, and dialysis purification. The 65:30:5 phosphatidylcholine:cholesterol:DSPE-PEG2000 molar composition was selected using literature-guided operational criteria; no formal Design of Experiments model was used. Particle size, PDI, zeta potential, dialysis-based apparent encapsulation efficiency, morphology, physicochemical properties, antibody integrity, HER2-binding activity, and 28-day storage stability were evaluated using DLS, microscopy, FTIR, DSC, TGA, XRD, SEC-HPLC, and HER2-binding ELISA.ResultsThe selected formulation had a mean particle size of 149.3 ± 5.8 nm, PDI of 0.22 ± 0.02, apparent zeta potential of +39.1 ± 2.1 mV under low-conductivity measurement conditions, and dialysis-based apparent encapsulation efficiency of 73.6 ± 3.7%. Recovered trastuzumab remained predominantly monomeric (94.9 ± 1.4%) and largely retained HER2-binding activity (92 ± 6%). Refrigerated samples showed the smallest 28-day changes, whereas 37 °C storage increased particle size and PDI and reduced apparent encapsulation efficiency.ConclusionsThe selected formulation met the predefined physicochemical and protein-quality endpoints and largely preserved trastuzumab integrity and HER2-binding activity. Refrigeration provided the greatest short-term stability. The findings establish an integrated characterization benchmark under the tested conditions but do not demonstrate quantified surface PEG density, physiological zeta-potential behavior, hemocompatibility, prolonged circulation, biodistribution, or therapeutic efficacy.