Characterization of Recombinant Human Lactoferrin Expressed in Komagataella Phaffii

Xiaoning Lu, Chad Cummings, Udodili A. Osuala, Neela H. Yennawar, Kevin E. W. Namitz, Brittney Hellner, Pamela B. Besada-Lombana, Ross D. Peterson, Anthony J. Clark

bioRxiv 2024.01.09.574900

DOI: https://doi.org/10.1101/2024.01.09.574900

ABSTRACT

We performed a thorough analysis and characterization of multiple batches of Helaina recombinant human lactoferrin (rhLF, Effera™) expressed at an industrial scale in a yeast system. Bottom-up LC-MS/MS-based proteomics analysis detected the full sequence of Helaina rhLF protein and confirmed that its amino acid sequence is identical to that of native human LF (Uniprot i.d. P02788). Helaina rhLF had a protein purity of 98% or higher as determined by three orthogonal methods; reversed-phase HPLC, SDS-PAGE, and LC-MS proteomics analysis. N-linked glycans were detected at three known glycosylation sites, namely, Asparagines-156, -497, and -642. The identified N-glycans of Helaina rhLF were predominantly oligomannose structures with five to nine mannoses (M5-M9), which we also report to be present in both the native human and bovine LF. human milk LF (hmLF) possessed lower levels of oligomannose structures and were mainly M5 and M6. Helaina rhLF protein secondary structure was nearly identical to that of hmLF, as revealed by microfluidic modulation spectroscopy. Results of small-angle X-ray scattering (SAXS) and analytical ultracentrifugation analyses confirmed that, like hmLF, Helaina rhLF displayed well-folded globular structures in solution. Reconstructed solvent envelopes of Helaina rhLF, obtained through the SAXS analysis, demonstrated a remarkable fit with the reported crystalline structure of iron-bound native hmLF. Differential scanning calorimetry investigations into the thermal stability of Helaina rhLF revealed two distinct denaturation temperatures at 68.7±0.9 °C and 91.9±0.5 °C, consistently mirroring denaturation temperatures observed for apo-and holo-hmLF. Overall, the characterization analysis results affirmed that Helaina rhLF was of high purity and exhibited globular structures closely akin to that of hmLF.

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