Abstract

Background: Brucellosis remains a significant zoonotic infection affecting approximately 500,000 people worldwide annually, with no licensed human vaccine available. Recombinant influenza virus vectors expressing Brucella spp. antigens represent a promising vaccine platform. However, transitioning from laboratory constructs to clinical candidates requires validation of scalable purification methods compliant with Good Manufacturing Practice (GMP) standards. This study aimed to develop and optimize a pilot-scale purification protocol for these vectors. Methods: Recombinant influenza A viruses (H5N1) expressing Brucella spp. antigens (Omp16, Omp19, L7/L12, Cu-Zn SOD) were propagated in MDCK cell culture. The optimized purification process included: (1) clarification; (2) ultrafiltration/diafiltration (100 kDa MWCO); (3) two-step chromatography (anion-exchange Q-Sepharose® Fast Flow and multimodal Capto™ Core 700); and (4) sterile filtration. Process validation was performed across three independent pilot-scale batches (20 L each). Results: The purification process demonstrated high reproducibility for all constructs. Final preparations met established quality criteria: infectious titer ≥ 5.2 log10 TCID50/mL, hemagglutination activity 7.33 ± 0.58–8.33 ± 0.58 log2, total protein content 157–305 μg/mL, residual host cell DNA < 10 ng/dose, and bacterial endotoxin levels ≤ 0.15 IU/mL. The overall recovery of infectious virus was 20–24%, an optimal value for multi-stage bioprocessing. Preservation of the target genetic insert was confirmed in all final preparations by PCR and sequencing. Conclusions: The developed integrated purification protocol yields vectors with high purification efficiency, preserving biological activity and meeting regulatory quality requirements for residual host cell DNA and endotoxins. The technological platform demonstrated versatility, robustness (inter-batch coefficient of variation for yield did not exceed 10–12%), and scalability, establishing a foundation for preclinical and clinical studies of candidate brucellosis vaccines.

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