Evaluation of Genetic Stability of Recombinant Human Factor VIII by Peptide Mapping and On-line Mass Spectrometric Analysis

Purpose. The genetic stability of a recombinant human factor VIII (rhFVIII) product expressed in Chinese hamster ovary cells (Recombinate) has been evaluated through comparisons of the protein produced at the beginning, middle and end of a typical production campaign.Methods. Recombinant human factor VIII was incubated with thrombin, the resulting four polypeptides were isolated by RP-HPLC, subjected to proteolysis with trypsin, and the peptide mixtures were resolved by RP-HPLC. Tryptic peptide mixtures were subjected to online mass spectrometric analysis using an electrospray ionization source interfaced to a quadrupole mass analyzer scanning from 1950–200 amu, and the peptide ion data were compared for three lots produced from the beginning, middle and end of a production campaign.Results. The UV elution profiles for each of the rhFVIIIa polypeptides were highly similar for factor VIII isolated from the beginning, middle and end of production. Total ion data from the peptide maps derived from three lots of rhFVIII were compared by MH¹⁺ values as a function of scan range. A total of 918 ions were analyzed for the four polypeptides of rhFVIII produced at the beginning, middle and end of a production campaign. The ions were detected at the same relative retention times, as indicated by the similar scan numbers for the three lots.Conclusions. These observations support that rhFVIII preparations produced from the beginning, middle and end of a production campaign were highly similar, and demonstrate genetic stability in the manufacturing process of Recombinate.