Virus-like particles (VLPs) are non-infective vaccine candidates that have gained interest given their natural ability to elicit strong immune responses. Particularly, HIV-1 Gag-based VLPs are one of the most described platforms for vaccine development, provided their ability for successful pseudotyping either by genetic engineering or click chemistry. When Gag polyprotein is recombinantly expressed, VLPs are naturally assembled in the vicinity of the cell membrane and then secreted by cell budding, taking part of the host cell membrane. Their properties are dependent upon the cell line and manufacturing method. Although great advancements toward the implementation of analytical methods have been made, VLP quality attributes are quite unclear whenever production is enhanced by metabolic engineering or process intensification strategies. This work offers a comparative study of VLP quality attributes upon transient gene expression (TGE) in HEK293 cell cultures operated in batch and perfusion mode. Moreover, the impact of specific productivity is also studied by ataxia telangiectasia mutated (ATM) gene silencing, which has been reported to enhance fourfold VLP production. A linear negative correlation was found between the ratio of Gag monomers/VLP and specific productivity. 3100 ± 100 monomers/VLP were obtained for the standard batch production, dropping to 1900 ± 100 and 800 ± 60 for the perfusion and batch ATM-knockdown conditions, respectively. Furthermore, functionalization rates were measured in terms of Cy5 per total particles (TP). Both perfusion-derived nanoparticles achieved functionalization rates of 2800 Cy5/TP. On the contrary, those nanoparticles produced in batch yielded functionalization rates below 1000 Cy5/TP. Moreover, a complete lipidome analysis revealed a relative decrease in the quantity of lipid/particle for all studied conditions in comparison to the standard batch production. Finally, all VLP samples were characterized to assess the impact of the differential physicochemical properties upon purification and stability rates. KEY POINTS: • VLP quality inversely correlates with Gag-specific productivity and operation mode. • Functionalization and lipid content drop with metabolic burden or ATM silencing. • Perfusion enables high VLP recovery and lyophilization with preserved morphology.