Lyophilized HIV‑1 Gag virus‑like particles: improved quality and stability

Virus-like particles (VLPs) have good potential for novel vaccine development. Particularly, HIV-1 Gag VLPs, present a robust platform for vaccine production. However, successful translation from the laboratory to biomanufacturing requires focus on formulation, with lyophilization playing a pivotal role in enhancing stability and facilitating distribution. The challenge lies in the potential damage inflicted during the lyophilization, impacting product integrity and stability. To address this, optimization of formulations and methodologies are imperative. Formulation excipients, play an important role, with cryo-lyo protectants, bulking agents, buffers, tonicity modifiers, and surfactants being commonly utilized. This work focuses on optimizing lyophilization formulation for Gag::eGFP VLPs, employing a Design of Experiments approach. The impact of five main excipients is studied, using size, concentration, and quality of the Gag::eGFP VLPs as attributes to characterize. Moreover, the impact of specific storage conditions after lyophilization is also evaluated. The lyophilized Gag::eGFP VLPs, formulated with the optimized formulation, exhibited characteristics akin to those observed in the non-lyophilized Gag::eGFP VLPs. The stability studies revealed the Gag::eGFP VLPs stored at 4 °C for 4 weeks, in lyophilized or reconstituted form maintain their integrity. While these results demonstrate short-term stability as a proof of concept, further studies are required to assess long-term shelf life. This work provides a systematic DoE-based framework for the optimization of lyophilized HIV-1 Gag VLP formulations and contributes new insights into the preservation of physicochemical quality attributes during freeze-drying and subsequent storage, supporting the future development of stable VLP-based vaccine platforms.