Determination of rFVII Concentration in Cell Culture Supernatant Using VIISelect Resin and RP-HPLC-UV

Title:Determination of rFVII Concentration in Cell Culture Supernatant Using VIISelect Resin and RP-HPLC-UV

Volume: 18 Issue: 10

Author(s): Mehdi Khodadadian*, Nahid Zarezadeh, Hossein Behrouz and Zeinab Ahsani

Affiliation:

• Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Iran

Keywords: rFVIIa, hemophilia, VIISelect, RP-HPLC, solid-phase extraction, biopharmaceutical.

Abstract:

Background: Recombinant activated human coagulation factor VIIa (rFVIIa), a vitamin K-dependent serine protease, was originally developed by Novo Nordisk® for the treatment of patients with FVII deficiency as well as patients with hemophilia A and B with inhibitors against FVIII or FIX. The gene for human FVII is cloned and expressed in baby hamster kidney (BHK) cells, and the protein is secreted into the culture media, which is then converted to the active form during chromatographic purification steps. When secreted into the culture media, measuring the concentration levels of FVII is needed in order to monitor, control and optimize the production processes. However, because of the high complexity of such media, reliable analytical techniques are required for this purpose.

Objective: This work focuses on the analytical application of VIISelect resin (GE Healthcare) as a highly selective adsorbent for cleanup and preconcentration of rFVII in culture supernatant before analysis by reversed-phase high-performance liquid chromatography (RP-HPLC).

Methods: Empty 1 ml SPE cartridges were packed with VIISelect resin. Four types of solutions were used for the sample preparation. The RP-HPLC separation was conducted on a C4 column with UV detection.

Results: The method shows recoveries greater than 97% in culture medium with relative standard deviations (RSDs) of less than 2%. The limits of detection and quantification were 0.039 mg/L and 0.12 mg/L, respectively.

Conclusion: The method showed better performance in terms of precision and accuracy for rFVIIa determination in cell culture supernatant compared to other techniques, like ELISA and SDSPAGE.

Cite this article as:

Khodadadian Mehdi*, Zarezadeh Nahid, Behrouz Hossein and Ahsani Zeinab, Determination of rFVII Concentration in Cell Culture Supernatant Using VIISelect Resin and RP-HPLC-UV, Current Pharmaceutical Analysis 2022; 18 (10) . https://dx.doi.org/10.2174/1573412918666220901155615