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.

Background: Therapeutic recombinant biological products are essential in modern medicine, but their immunogenicity remains a major concern for both clinical safety and therapeutic efficacy. Protein aggregation may occur during manufacturing, storage, transportation, and administration, potentially increasing immune recognition. Here, we have reviewed the mechanisms underlying aggregation-associated immunogenicity and summarized strategies for mitigating the associated risks. Methods: We integrated published evidence on therapeutic recombinant proteins, monoclonal antibodies, antibody–drug conjugates, peptide and glycoprotein hormones, and recombinant blood products. We focused on aggregation-induced structural changes, immune activation pathways, differences among product types and aggregation states, and approaches for controlling aggregation and mitigating immunogenicity. Results: Protein aggregation creates or reveals neoepitopes by disrupting the native conformational state, exposing previously buried hydrophobic regions, and generating nonnative interfaces. Aggregates may further promote innate and adaptive immune responses through repetitive epitope presentation, pattern-recognition receptor signaling, complement activation, cytokine release, dendritic-cell maturation, enhanced antigen presentation, and B-cell receptor cross-linking, ultimately increasing the likelihood of anti-drug antibody formation. Aggregate size and morphology, protein characteristics, formulation, storage conditions, administration route, and other biological factors influence the immunogenic potential of aggregates. Thus, effective risk reduction requires integrated control strategies, including molecular and structural optimization, formulation design, manufacturing-process control, appropriate storage and transportation, and sensitive analytical monitoring. Emerging artificial-intelligence approaches may further facilitate the prediction and design of low-immunogenicity biologics. Conclusions: Protein aggregation is a multifactorial determinant of immunogenicity. Controlling aggregation throughout the product lifecycle is essential to reduce immune-mediated risks and maintain safety, stability, and efficacy of therapeutic recombinant biological products.