Optimization of methods for isolation and purification of outer membrane vesicles (OMVs) from Neisseria lactamica.

Outer membrane vesicles (OMVs) are nanoparticles released by Gram-negative bacteria during growth, mainly under stress conditions. OMV-based vaccines have played an important role in vaccination against Neisseria meningitidis serogroup B (MenB), stimulating research into novel approaches for developing more effective vaccines. OMVs released by the bacterium Neisseria lactamica have emerged as a promising platform for new vaccine development, especially as carriers in subunit vaccines. Despite their importance, some challenges remain in obtaining and purifying OMVs. The most commonly employed methods for OMV isolation and purification are ultracentrifugation (UC) and size exclusion chromatography (SEC). However, these techniques could present limitations for large-scale production and often result in low yields. This study investigated techniques such as tangential flow filtration (TFF), membrane chromatography, and mixed-mode (multimodal) chromatography as potential replacements for UC and SEC. Among the TFF methods evaluated, the sample obtained on the membrane with a 300-kDa cutoff showed a profile more similar to UC but with more than double the total protein recovery. Sartobind® Q membrane chromatography was ineffective for OMV purification, in the conditions evaluated, with a recovery of 8.7%. Conversely, multimodal Capto™ Adhere chromatography recovered 59.0%, while Capto™ Core 400 yielded a recovery of 72.0%, proving to be more effective for purification when analyzed by high-performance liquid chromatography (HPLC). Thus, combining TFF with a 300-kDa membrane followed by Capto™ Core 400 chromatography can be applied as strategy for large-scale applications offering high recovery and purity. KEY POINTS: • Evaluation of TFF, membrane and multimodal chromatography techniques for OMV purification. • Improved Neisseria lactamica OMV yields combining TFF and multimodal chromatography. • A process for OMV purification from a non-pathogenic organism feasible to scale up.