Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB
Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB

Recombinant production of blood clotting factor VIIa as a biosimilar

A biosimilar (biogeneric) is a drug that has been produced via biotechnology and which has the same qualitative and quantitative composition and the same administrative form as the reference drug. Its bioequivalence to the reference drug must be confirmed by suitable studies. At the Fraunhofer IGB, we have established a generic procedure for clotting Factor VII on behalf of the Iranian company, CinnaGen Biopharma.

Blood coagulation is a complex process in which different clotting factors trigger a cascade of enzymatic reactions to finally form a stable fibrin clot. If clotting factors such as Factor VII are missing within this chain, the formation of the fibrin network is prevented. In affected persons this leads to a coagulation disorder, namely hemophilia. The frequency of such a disease at 1 in 500,000 is quite rare. Treatment consists of the intravenous application of (recombinant) activated Factor VII produced by genetic engineering or Factor VII which is obtained from human blood.

Recombinant production in mammalian cells

Because of the risk of viral contamination and the limited availability of banked human blood, the blood coagulation proteins previously isolated from human blood plasma are with increasing frequency being produced by genetic engineering. Human factor VII is a vitamin K-dependent serine protease, consisting of a single-chain molecule of 406 amino acids. During the coagulation cascade it is transformed into the active two-chain form, FVIIa.

To establish the new synthetic pathway, we have cloned the DNA of the protein in a specific vector system, which was transfected in a stable manner into Baby Hamster Kidney Cells (BHK cells) and amplified a high number of copies. The cells are adapted to suspension culture conditions in a serum-free medium containing also little protein to facilitate production as efficient as possible. Protein FVII is secreted into the medium as a pro-enzyme (zymogen); this makes processing significantly easier. Transformation to the active enzyme occurs quantitatively during protein purification in a later stage of production. Thus, the final product in this production process is the activated Factor VII (rFVIIa).

High factor VII expression in the production cell line

Phase contrast absorption of the adherent growing BHK cell line.
Phase contrast absorption of the adherent growing BHK cell line (ATCC CRL 10314, left) and adapted to a serum-free and low protein medium as well as suspension conditions (right).

This research was able to establish a high expression cell clone (BHK cell line) with an expression rate of 4.9 µg/ml (Table). The quantitative analysis of factor VII in the culture medium supernatant of the production cell line was measured by ELISA. The biological activity was verified with the COASET FVII (CHROMOGENIX, Co.) assay and the clotting assay of Dade Behring, Co. The western blot shows a specific band of the Factor VII protein from a sample of the non-concentrated and the 10-fold concentrated culture medium supernatant. Serum-free medium was tested as a control.

  ELISA Chromogener
substrat assay		 Clotting assay
BHK-Production cell line 4,5 µg/ml 4,5 µg/ml

verified

Table: Quantitative analysis and determination of the biological activity of factor VII in the culture medium supernatant of the BHK production cell line by means of ELISA, the chromogenic substrate assay and the clotting assay

Outlook

At Fraunhofer IGB, it was possible to establish an additional successful production process for a biogeneric with the production of clotting factor VII. This consists of the production of DNA constructs, cloning and the stable transfection into different expression systems, cultivation also of larger volumes and the final purification of the active protein (downstream processing). The partner company, CinnaGen Biopharma, will shortly apply to the competent authority for a drug approval