Valuation of an N-glycosylation profile of recombinant blood coagulation factor VIIa molecule

Introduction. The specific activity of blood coagulation factor VIIa depends, among other things, on the presence of N-acetylneuraminic acid (sialic acid) and phosphate residues in the molecule. Thus the authenticity of glycosylation profile as one efficacy and safety proofs of drug product is of particular interest. However, the whole variability of posttranslational modifications requires an individual approach to corresponding assessment processed in accordance with the requirements and recommendations of the comparison of biosimilar drug products. Methods. Objects under the study referred as: original drug product NovoSeven, biosimilar drug product Coagil-VII and European reference standard of Human Coagulation Factor VIIa (rDNA) CRS. Two methodological approaches of high-pH-anion-exchange chromatography (HPAEC) and hydrophilic interaction liquid chromatography (HILIC) with fluorescent and mass-spectrometric detection were used for the analysis of oligosaccharide distribution in all factor VIIa molecules. Results. А set of rFVIIa modifications represented basically by complex biantennary oligosaccharides with insignificant part of triantennary structures was established in this study. The most characteristic modification for Coagil-VII biosimilar was complex bisialylated core-fucosylated biantennary group. Conclusion. The evaluation of the N-glycosylation profile of recombinant blood coagulation factor VIIa based on the comparative analysis of biosimilar drug product, original drug product, and CRS standard sample revealed difference in quantitation of N-glycans among with general matching of the majority of peak retention times. Mass-spectrometric detection allowed to carry out a structural interpretation of HPLC data. A biosimilar drug product was used as an example to illustrate the necessity for factory standards certified in a prescribed manner for the further glycan profile authentication.

Eptacog alpha, glycosylation profile, authenticity, mass spectrometric analysis, standard sample