Post-translational modification fidelity of recombinant human lactopontin expressed in Kluyveromyces lactis

Recombinant human lactopontin (rhLPN), an equivalent of human milk lactopontin, is of increasing interest for human nutrition applications due to its roles in mineral binding, gastrointestinal function and immune modulation. These properties depend strongly on post-translational modifications, particularly phosphorylation and glycosylation. Here, we present a comprehensive molecular comparison between rhLPN produced in Kluyveromyces lactis at laboratory and pilot scale with native human lactopontin (nhLPN) isolated from human milk. Mass spectrometry-based peptide mapping confirmed the primary structure and identified extensive phosphorylation, consistent with the native protein. Middle-up analyses demonstrated closely matched phosphoform distributions between rhLPN and nhLPN, while glycosylation profiling revealed a defined population of low-complexity O-glycoforms localized to the N-terminus. Functional assessment demonstrated substantially greater iron binding by phosphorylated rhLPN compared with dephosphorylated and non-phosphorylated forms. Similar phosphorylation-dependent behaviour was observed for bovine lactopontin, supporting a conserved role for phosphorylation in mineral interaction. Across five 750 L pilot scale batches, both phosphorylation and glycoform distributions were highly consistent, indicating robust process reproducibility. Together, these results demonstrate that rhLPN produced in K. lactis recapitulates key structural and functional attributes of nhLPN, supporting its suitability as a scalable ingredient for nutrition applications.