The development of industry standard sample qualification procedure of recombinant activated blood clotting factor VII for proving of identity by peptide mapping method

Introduction. The use of European reference standard with the known structure (CRS Human coagulation factor) as a reference allows to confirm identity of the primary structure of new batches of the recombinant protein. Manufactures normally use an industry standard sample due to the high cost of the European reference standard and to make sure that national samples are independent from the international reference standards. Regulatory requirements for the qualification procedure of an industry standard sample are absent. Materials and methods. Batch of recombinant activated clotting factor VII (rFVIIa) (Eptacog alpha) ("Koagil") was selected as a candidate material. The method of strong hydrolysis with the initial deglycosylation of the molecule was used. Amino acid sequence of rFVIIa, described in the monograph 01/2015:2534 of European Pharmacopoeia (Ed. 9.0) was used as a reference. Peptide mapping and mass-spectrometric assay were used in the study. Results. Qualification procedure of industry standard sample of rFVIIa was developed. The standard sample was used for the proof of identity of rFVIIa by RP-HPLC peptide mapping. Qualification procedure of an industry standard sample of rFVIIa allows to obtain fully characterized reference sample with the established structure. The use of this standard sample guarantees accurate assessment of the identification by peptide mapping of the new batches of rFVIIa with or without European reference standard. Conclusion. Peptide mapping and mass-spectrometric assay allows us to confirm amino acid sequence of light and heavy chains of rFVIIa with 100 % coverage. The peptide map was established for the candidate material in comparison with the original drug NovoSeven and the selection of characteristic peaks was justified. These peaks were characterized by stability and symmetry factors. Amino acid sequence of peptides corresponding to the reference peaks and their primary localization in the molecule were established by the use of tandem mass-spectrometry.