Glycosylation-based distinction of endogenous and recombinant complement factor H enables selective quantification in human plasma.
TL;DR
A generalizable approach for identifying molecular differences between endogenous and recombinant proteins and leveraging these features to develop high-quality bioanalytical assays is outlined.
Abstract
Background
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. RESEARCH
Design
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.
Results
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.
Conclusions
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.