Transient Expression Of Recombinant SPDGFRα-Fc In CHO DG44 Cells Using Orbitally Shaking Disposable Bioreactors

In this study, we used sPDGFR-Fc as a model protein, new orbital shaking disposable bioreactor and gene transient transfection technology to study how to preparate antibodies and antibody-like protein drug candidates in the mammalian cells technical system. First, we do a preliminary research about CHO DG44 cell culture technology. CHO DG44 cells were cultured in 50mL bioreactor which fixed in the orbital shaking shaker. Cell growth curve was made based on growth state of CHO DG44 cells. Second, Transient transfection technology of CHO DG44 cells was developed. With classic plasmid pEGFP-N1 as vector and straight-chain PEI (25KD) as transfection reagent, we builded a mature recombinant protein expression technology about CHO DG44 cells. A maximal cell density of up to 3.3×106cells/mL was obtained in the shaking bioreactors. It was observed that maximal GFP expression was reached at DNA:PEI ratios of 1:4 and 1:5. In this condition,41.51±3.28%cells were transfected and able to express GFP protein.Four gene expression vector about PDGFR which include PCDNA3.1-PDGFR-FC, pIRESneo3-EGFP-PDGFR-FC, pIRESneo3-KH2-PDGFR-FC and pIRESneo3-KH PDGFR-FC were constructed. Transfection were done to selected the best plasmid to do the following research which included western-blotting to detect the target protein, ELISA to check the protein expression. The sPDGFRa-Fc fusion protein was transiently expressed in CHO DG44 cells in 50-mL orbital shaking bioreactors. sPDGFRa-Fc was expressed as a dimeric protein with potential glycosylation. The final yield of sPDGFRa-Fc in the culture supernatant was as high as 16.68 mg/L.In addition, we also pay our attention on the transient transfection mechanism of DG44 mainly through Malvern particle analyzer and observation the realtonship between ion concentration and complex stability. The results showed that salt ion plays a key role in DNA-PEI complex formation and stability.In the case of no salt ions, complex formation of the particles will be unstable. As the time goes on, the particle size has been larger within 8 hours. Diameter of complex was up to 4206nm in the salt solution. In basic medium, the particles whose size is about 800nm remain relatively stable in 30min due to some complex components of the medium.