en بیوتکنولوژی Biotechnology پژوهشي Research <strong>Background:</strong> <a name="_Hlk535574491"></a><a name="_Hlk535577862">Pilot and large-scale production of recombinant proteins require the presence of stable clones</a>, but the process of selecting stable clones is time consuming. Moreover, continuous clone culturing in large-scale production may cause loss of incoming plasmid and recombinant genes.<br> Considering the advancements in Transient Gene Expression (TGE) technology, the large-scale expression of factor IX (FIX) was investigated in HEK cells by the TGE technique.<br> <strong>Materials and methods:</strong> HEK cells were seeded in a cell factory, and then transfected by pcDNA-hFIX plasmid using calcium phosphate co-precipitation method. Stable HEK-hFIX cells were also seeded in a cell factory, separately. After adding vitamin K, recombinant FIX was quantified in conditioned media using an ELISA. Moreover, its functional activity was assayed using an aPTT test.<br> <strong>Results:</strong> The results showed that the expression and activity of FIX by TGE technology was, respectively, 1.6 and 1.5 times higher than that obtained through stable HEK-FIX cells. Since calculating the specific activity revealed that for all time periods it is 0.2 mU/ng, so the increase in activity is due to the increase in the amount of FIX.<br> <strong>Conclusions:</strong> HEK cells with higher transfectability seemed to be an appropriate alternative for transient expression for large-scale protein production. Furthermore, if rapid expression of recombinant proteins is intended, TGE can replace costly and low-yield methods.<br> &nbsp; Coagulation factor IX, large-scale production, Transient Gene Expression technology 29 35 http://rmm.mazums.ac.ir/browse.php?a_code=A-10-1050-1&slc_lang=en&sid=1 Jafar Vatandoost جعفر وطن دوست j.vatan@hsu.ac.ir 1 10031947532846007754 Yes Department of Biology, Hakim Sabzevari University, Sabzevar دانشگاه حکیم سبزواری Mohammad amin Azimifar محمدامین عظیم فر aminazimifar@gmail.com 2 10031947532846007755 No Department of Biotechnology, Sabzevar Branch, Islamic Azad University, Sabzevar, Iran دانشگاه آزاد سبزوار