PhiC31 integrase can improve the efficiency of different construct designs for monoclonal antibody expression in CHO cells

• Published in: Protein expression and purification Vol. 134; pp. 89 - 95
• Main Authors: Ahmadi, Maryam, Mahboudi, Fereidoun, Ahmadi, Samira, Ebadat, Saeedeh, Nematpour, Fatemeh, Akbari Eidgahi, Mohammad Reza, Davami, Fatemeh
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.06.2017
• Subjects: Animals; Antibodies, Monoclonal - biosynthesis; Antibodies, Monoclonal - genetics; Attachment Sites, Microbiological; Chinese hamster ovary (CHO); CHO Cells; Cricetinae; Cricetulus; Dual promoter vector; Gene Expression; Genetic Vectors - genetics; Integrases - biosynthesis; Integrases - genetics; PhiC31; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Site-specific recombination; Transgenes; Dual promoter vector; Chinese hamster ovary (CHO); PhiC31; Site-specific recombination
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2017.04.005

Several types of expression vectors have been used for recombinant protein expression in Chinese hamster ovary cells (CHO) which usually result in variable and unstable levels of expression. In this study, we have compared the mAb0014 expression level of single ORF/IRES vector and dual ORF vector in the presence and absence of phiC31 integrase targeting system. Both expression vectors contain an elongation factor 1α (EF1α) promoter upstream of LC and harboring an attB site. CHO-S cells were co-transfected with single ORF/IRES or dual ORF vectors along with a phiC31 integrase expression vector which can catalyze recombination between attB site and pseudo-attP sites presented in the mammalian genome. Our results demonstrated that dual ORF vector in the presence of phiC31 integrase expression vectors (+FC31 2P) generated more recombinant antibody in comparison to its negative control (-FC31 2P). Moreover, both of +FC31 2P and -FC31 2P cell pools yield higher recombinant protein in comparison to single ORF/IRES vector (FC31 IRES) cell pools. Stability of expression in phiC31 co-transfected cell pools (+FC31 2P and +FC31 IRES) had no considerable changes. Our results indicated that the dual ORF vector using integrase can support the generation of cell lines with stable transgene expression at an elevated mAb relative to single ORF/IRES vector. •PhiC31 integrase-mediated gene targeting can increase biopharmaceutical protein titer.•At least 25 fold higher amounts of antibody.•Up to 100 fold in optimum ratio of phiC31 integrase helper vector.•PhiC31 integrase-mediated gene targeting can decrease the required time to achieve sufficient amounts of protein.