Protein & Peptide Letters, 2010, 17, 919-924 919 0929-8665/10 $55.00+.00 © 2010 Bentham Science Publishers Ltd. Transient Expression of Recombinant sPDGFR-Fc in CHO DG44 Cells using 50-mL Orbitally Shaking Disposable Bioreactors Yun-Xia Sang 1,# , Xiao-Wei Zhang 1,2,# , Xiao-Jia Chen 3 , Kui Xie 1 , Chui-Wen Qian 1 , An Hong 3 , Qiu-Ling Xie 3,* and Sheng Xiong 1,* 1 Biomedical R&D Center, 3 Institute of Bioengineering, Guangdong Provincial Key Laboratory of Bioengineering Medi- cine, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou 510632, Guangdong, P.R. China; 2 College of Translational Medicine, Nanchang University, Nanchang 330031, Jiangxi, P.R. China Abstract: Overactivity of platelet-derived growth factor (PDGF) has been linked to malignant cancers. High levels of PDGF result in the activation of its receptors (PDGFRs) and the over-proliferation of cells. Therefore, interfering with this signaling pathway in cancer cells could be significant for anti-cancer drug development. In a previous study, the sPDGFR-Fc fusion protein expressed in static CHO-k 1 cells showed an anti-proliferative effect on vascular endothelial cells. However, it was difficult to obtain a large quantity of this fusion protein for further functional studies. In the present study, the sPDGFR-Fc fusion protein was transiently expressed in Chinese Hamster Ovary (CHO) DG44 cells in 50-mL orbital shaking bioreactors. sPDGFR-Fc was expressed as a 250-kDa dimeric protein with potential glycosylation. The final yield of sPDGFR-Fc in the culture supernatant was as high as 16.68 mg/L. Our results suggest that transient expres- sion in orbital shaking bioreactors may be feasible for preparation of recombinant proteins used for preclinical studies. Keywords: PDGF, soluble PDGFR-Fc, transient gene expression, CHO DG44 cells, Orbitally shaking bioreactor, disposable bioreactor. INTRODUCTION Human cancers over-express a number of growth factors and their receptors, such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) [1, 2]. Therefore, inhibitors of these signaling pathways are potential anti-cancer drugs [3, 4]. Among these signaling inhibitors, recombinant soluble growth factor re- ceptors are potential specific anti-cancer drugs. PDGF-AA and PDGF-BB are generally over-expressed in lymph node metastatic tumors and pancreatic cancers [5]. Since the PDGF signaling pathway is involved in cell prolif- eration, blocking the interaction between PDGF and PDGFR could be a potential anti-cancer approach [3]. PDGFR has two isoforms that dimerize upon binding the PDGF dimer, leading to three possible receptor combinations, namely -, - and – [6]. The  isoform binds both the A and B- forms of PDGF, whereas the -isoform only binds PDGF- BB [7]. In a previous study, the soluble extracellular domain of the -isoform of PDGFR (sPDGFR) was fused to the Fc fragment of human immunoglobulin IgG, and the resultant fusion protein (sPDGFR-Fc) was expressed in static- cultured Chinese hamster ovary (CHO) cells. The fusion *Address correspondence to these authors at the Biomedical R&D Center, 5/F Building of Biology, Jinan University, Guangzhou 510632, Guangdong, P.R. China; Tel: +86-20-85220504-311; Fax: +86-20-85220504-309; E-mail: xiongsheng@jnu.edu.cn and, Institute of Bioengineering, 7/F Build- ing of Biology, Jinan University, Guangzhou 510632, Guangdong, P.R. China; Tel/Fax: +86-20-85221983; E-mail: txql@jnu.edu.cn # These authors contributed equally. sPDGFR-Fc protein was able to competitively bind both A- and B-forms of PDGF and to inhibit cell proliferation [8]. The Fc-fusion not only stabilized sPDGFR but also facili- tated purification. At present, most therapeutic proteins from mammalian expression systems are produced in stably transfected CHO cells [9]. However, in “classical” approaches of protein ex- pression from stable mammalian cells, it typically takes months to establish a productive host cell line. In the last decade, scalable transient gene expression (TGE) using sus- pension cultures of transfected mammalian cells has also been shown to rapidly generate gram-quantities of recombi- nant proteins for biochemical and preclinical studies [10-17]. The culture of suspended mammalian cells in orbitally shak- ing bioreactors is a preferred method for TGE due to its high mass transfers, low shear force, excellent mixing capacity, low cost and scalability [18]. The development of dispos- able-material based bioprocesses and the use of orbitally shaking bioreactor systems are aimed at simplifying the technology for the production of biopharmaceuticals. Orbi- tally shaking disposable bioreactor units up to 1500 L in vol- ume have been tested for mammalian cell culture. Coupling the advantages of TGE and orbitally shaking the cell-culture at the 100-L scale, hundreds of milligrams or even gram amounts of a monoclonal antibody (IgG) are produced using suspension-adapted HEK 293 EBNA SF cells and NSO cells [19]. In this study, we recombinantly expressed sPDGFR-Fc in CHO DG44 cells with transient transfection in an orbitally shaking disposable bioreactor (tubespin®, TPP Trasadingen, Switzerland). Cell density and DNA:PEI ratio were varied to