title: Enhancement of transient gene expression and culture viability using Chinese hamster ovary cells overexpressing Bcl-x(L).
abstract: Transient gene expression (TGE) provides a method for quickly delivering protein for research using mammalian cells. While high levels of recombinant proteins have been produced in TGE experiments in HEK 293 cells, TGE efforts in the commercially prominent CHO cell line still suffer from inadequate protein yields. Here, we describe a cell-engineering strategy to improve transient production of proteins using CHO cells. CHO-DG44 cells were engineered to overexpress the anti-apoptotic protein Bcl-x(L) and transiently transfected using polyethylenimine (PEI) in serum-free media. Pools and cell lines stably expressing Bcl-x(L) showed enhanced viable cell density and increased production of a glycosylated, therapeutic fusion protein in shake flask TGE studies. The improved cell lines showed fusion protein production levels ranging from 12.6 to 27.0 mg/L in the supernatant compared to the control cultures which produced 6.3-7.3 mg/L, representing a 70-270% increase in yield after 14 days of fed-batch culture. All Bcl-xL-expressing cell lines also exhibited an increase in specific productivity during the first 8 days of culture. In addition to increased production, Bcl-x(L) cell lines maintained viabilities above 90% and less apoptosis compared to the DG44 host which had viabilities below 60% after 14 days. Product quality was comparable between a Bcl-xL-engineered cell line and the CHO host. The work presented here provides the foundation for using anti-apoptosis engineered CHO cell lines for increased production of therapeutic proteins in TGE applications.
authors: Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, 3400 North Charles Street, 221 Maryland Hall, Baltimore, Maryland 21218-2694, USA.
authors: Brian S
authors: BS
authors: Majors
authors: Michael J
authors: MJ
authors: Betenbaugh
authors: Nels E
authors: NE
authors: Pederson
authors: Gisela G
authors: GG
authors: Chiang
mesh_terms: D000818:Animals; D017209:Apoptosis; D016466:CHO Cells; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D015870:Gene Expression; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D014162:Transfection; D051020:bcl-X Protein / Q000235:genetics*
chemical_list: D011993:Recombinant Fusion Proteins; D051020:bcl-X Protein