Structure and function of fermentation-derived bovine lactoferrin produced from Komagataella phaffii

Area of Science:

• Biotechnology
• Biochemistry
• Food Science

Background:

• Bovine lactoferrin (bLf) possesses significant health benefits but is limited by low natural abundance and high production costs.
• Precision fermentation presents a viable method for producing recombinant bovine lactoferrin (rbLf), enhancing its availability for diverse applications.

Purpose of the Study:

• To confirm the structural and functional similarity of rbLf, produced in *Komagataella phaffii*, to native bLf for regulatory compliance and market expansion.
• To validate rbLf as a biosimilar alternative to native bLf, ensuring comparable efficacy and safety.

Main Methods:

• Intact mass analysis and LC-MS N-linked glycan profiling were employed to compare the molecular weight and glycosylation patterns of rbLf and bLf.
• Isoelectric point and core amino acid sequence analysis confirmed the identity between rbLf and bLf.
• Functional assays assessed rbLf's iron-binding capacity, receptor interactions, epithelial cell growth promotion, antimicrobial activity against *Escherichia coli*, lipopolysaccharide (LPS) binding, and LPS-induced toll-like receptor 4 antagonism.

Main Results:

• rbLf exhibited a molecular weight (84 kDa) comparable to bLf (82-83 kDa) and shared identical isoelectric points and core amino acid sequences.
• Glycan profiling indicated predominantly high-mannose-based glycans on rbLf, similar to a significant portion of bLf glycans.
• rbLf demonstrated retained functional activities, including iron binding, epithelial cell growth promotion, *E. coli* inhibition, LPS binding, and antagonism of LPS-induced TLR4 activity, with statistically significant results (P < 0.0001 to P < 0.001).

Conclusions:

• The structural and functional data strongly support the substantial similarity between rbLf and native bLf.
• Biosimilar rbLf produced via precision fermentation is a viable, high-value ingredient with potential for expanded use in infant nutrition and adult health products.
• This research validates rbLf as a promising alternative to native bLf, paving the way for increased market accessibility and application.