Aggregation of therapeutic proteins can result from a number of stress conditions encountered during their manufacture, transportation, and storage. This work shows the effects of two interrelated sources of protein aggregation: the chemistry and structure of the surface of the container in which the protein is stored, and mechanical shocks that may result from handling of the formulation. How different mechanical stress conditions (dropping, tumbling, and agitation) and container surface passivation affect the stability of solutions of intravenous immunoglobulin are investigated. Application of mechanical shock causes cavitation to occur in the protein solution, followed by bubble collapse and the formation of high‐velocity fluid microjets that impinged on container surfaces, leading to particle formation. Cavitation was observed after dropping of vials from heights as low as 5 cm, but polyethylene glycol (PEG) grafting provided temporary protection against drop‐induced cavitation. PEG treatment of the vial surface reduced the formation of protein aggregates after repeated dropping events, most likely by reducing protein adsorption to container surfaces. These studies enable the development of new coatings and surface chemistries that can reduce the particulate formation induced by surface adsorption and/or mechanical shock.

Background/Objectives: Virus-like particles (VLPs) are effective vaccine platforms but are susceptible to degradation, which compromises stability and immunogenicity. A key challenge is the lack of sensitive early indicators of instability. This study aimed to systematically evaluate the stability of an aluminum-free recombinant hepatitis E virus VLP vaccine under various stresses and identify predictive markers of instability. Methods: The VLP vaccine was subjected to thermal stress (4 °C, 25 °C, 37 °C, 56 °C for up to 28 d), repeated freeze–thaw cycles (up to 30 cycles), and mechanical agitation (orbital shaking at 100 and 300 rpm for up to 12 d). Stability was assessed using a multi-parameter panel monitoring critical quality attributes: conformational and colloidal stability, formation of high-molecular-weight species, mean particle size, polydispersity index, charge heterogeneity, and antigen content. Results: Changes in charge heterogeneity were the earliest indicator of instability, detectable within 3 days at 25 °C, 8 h at 37 °C, and 4 h at 56 °C, preceding losses in structural integrity or antigen-binding function. The VLPs remained stable at 25 °C for 28 d. Freeze–thaw cycles induced a basic shift in charge variants without compromising structure or function, while high-intensity shaking (300 rpm) caused aggregation after 3–6 d. The effects of common excipients were also characterized. Conclusions: Charge-variant analysis serves as a sensitive and predictive marker for VLP vaccine instability. The study delineates the distinct impacts of different stress factors and provides critical data for optimizing formulation design and storage strategies to enhance VLP vaccine stability.

Background/Objectives: The rapid development of biopharmaceuticals has heightened attention from both industry and regulatory agencies toward product quality, particularly regarding subvisible particles as a critical quality attribute. Existing pharmacopoeial methods, Light Obscuration (LO) and Microscopic Particle Count (MC), exhibit limitations in meeting increasingly refined analytical requirements. Flow Imaging Microscopy (FIM) technology shows promise as an alternative, yet its standardized methodologies are still under development. Methods: This study employed polystyrene microsphere standard beads and intravenous immunoglobulin to perform instrument standardization and consistency evaluations on FIM instruments sharing the same operating principles but from different manufacturers. The consistency and transferability of particle counting across platforms were assessed. Additionally, particle images obtained from parallel testing on two platforms were classified using confusion matrices based on convolutional neural networks and the Unified Manifold Approximation and Projection (UMAP) dimensionality reduction method. Results: This study investigated the consistency and developed a transfer strategy for particle counting results across different FIM platforms. Analysis of particle image classification confirmed the consistency of image-based categorization while also revealing the complexity associated with cross-platform image recognition. Conclusions: The findings provide valuable insights for the further standardization of Flow Imaging Microscopy, supporting its potential as a reliable analytical tool for subvisible particle analysis in biopharmaceutical quality control.