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oai:figshare.com:article/34048967 2026-10-01T17:40:40Z category_4 category_13 category_14 category_19 category_21 category_873 category_46 category_734 category_64 portal_87 item_type_6 month_year_10_2026

Glycosylation-based distinction of endogenous and recombinant complement factor H enables selective quantification in human plasma Alexander Schäfer (193561) Paulina Dabrowska-Schlepp (18533925) Andreas Busch (5636033) Jian-You Chen (4672165) Michael Wiesener (6691424) Christoph Siethoff (1738063) Biochemistry Genetics Molecular Biology Pharmacology Biotechnology Chemical Sciences not elsewhere classified Immunology Biological Sciences not elsewhere classified Cancer HPLC-MS/MS bioanalysis C3 glomerulopathy targeted protein quantification affinity capture glycosylation navigo proteins glycopeptides <p>Therapeutic proteins constitute an important class of biologics, including recombinant coagulation factors, enzyme-replacement therapies and hormones. Generally, a high degree of structural similarity between endogenous and therapeutic proteins is essential. From a pharmacokinetic perspective, however, it is advantageous to selectively quantify the exogenous protein.</p> <p>This study presents a strategy for the specific quantification of CPV 104, a recombinant form of complement factor H (FH), in human plasma. Because CPV 104 is identical in amino acid sequence to endogenous FH, differential HPLC-MS/MS analysis was used to identify structural features that discriminate both forms.</p> <p>Differential analysis revealed unique glycosylation patterns on CPV 104, enabling development of a targeted HPLC-MS/MS assay for CPV 104-specific glycopeptides. Assay robustness was further enhanced by incorporating CPV 104 with stable isotope labeled glycan residues as an internal standard. High-throughput analysis was achieved through integration with an affinity enrichment step using a custom FH specific resin, supporting 96 well processing. The method was fully validated and demonstrated excellent specificity, precision, and accuracy. The validated assay was applied to a first in human pharmacokinetics study while simultaneously monitoring endogenous FH.</p> <p>This work outlines a generalizable approach for identifying molecular differences between endogenous and recombinant proteins and leveraging these features to develop high-quality bioanalytical assays.</p> 2026-10-01T17:40:40Z Text Journal contribution 10.6084/m9.figshare.34048967.v1 https://figshare.com/articles/journal_contribution/Glycosylation-based_distinction_of_endogenous_and_recombinant_complement_factor_H_enables_selective_quantification_in_human_plasma/34048967 CC BY 4.0