Structure and function of fermentation-derived bovine lactoferrin produced from <i>Komagataella phaffii</i>

Bovine lactoferrin (bLf) confers significant functional benefits for human health, but low concentrations in milk and high cost of commercial production limit availability and thus product application. Precision fermentation offers a solution to increase availability of biosimilar recombinant bLf (rbLf) thereby opening new opportunities for this high-value ingredient. To comply with regulatory requirements, we aimed to establish that rbLf from <i>Komagataella phaffii</i> is substantially similar to native bLf in structure and key functions. Intact mass analysis showed a molecular weight of 84 kDa for rbLf, comparable to 82-83 kDa of bLf. LC-MS <i>N</i>-linked glycan profiling revealed predominantly high-mannose-based glycans on rbLf, similar to ∼50% of bLf glycans. The isoelectric point and core amino acid sequence of rbLf and bLf are identical. rbLf retains the functional ability to bind and release iron, bind to intestinal Lf receptors, increase epithelial cell growth (>120% of control, <i>P</i> < 0.0001), reduce enteropathogenic <i>Escherichia coli</i> growth (>50% reduction, <i>P</i> < 0.0001), bind lipopolysaccharide (LPS) (+4-fold, <i>P</i> < 0.001), and antagonize LPS-induced toll-like receptor 4 activity (>40% reduction, <i>P</i> < 0.0001). These results demonstrate similarity of rbLf in structure and function to native bLf, supporting the effective application for expanded market opportunities for infant and adult health.