IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines.
A Tricistronic vector utilizing internal ribosome entry site (IRES) elements to express the light chain (LC), heavy chain (HC), and a neomycin phosphotransferase (NPT) selection marker from one transcript is designed for generation of mAb expressing CHO cell lines. As compared to the commonly used vectors, benefits of this design include: (1) minimized non-expressing clones, (2) enhanced stable mAb productivity without gene amplification, (3) control of LC and HC expression at defined ratios, and (4) consistent product quality. After optimization of the LC and HC arrangement and increasing selection stringency by weakening the NPT selection marker, this Tricistronic vector is able to generate stably transfected pools with specific productivity (qmAb) greater than 5pg/cell/day (pcd) and titers over 150mg/L. 5% of clones from these pools have qmAb greater than 20pcd and titers ranging from 300 to more than 500mg/L under non-optimized shake flask batch cultures using commercially available protein-free medium. The mAb produced by these clones have low aggregation and consistent glycosylation profiles. The entire process of transfection to high-expressing clones requires only 6 months. The IRES-mediated Tricistronic vector provides an attractive alternative to commonly used vectors for fast generation of mAb CHO cell lines with high productivity.
Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, #06-01 Centros, Singapore 138668, Singapore.
Steven C L
SC
Ho
Muriel
M
Bardor
Huatao
H
Feng
Mariati
Yen Wah
YW
Tong
Zhiwei
Z
Song
Miranda G S
MG
Yap
Yuansheng
Y
Yang
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016466:CHO Cells; D002470:Cell Survival; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D005822:Genetic Vectors / Q000235:genetics*; D006031:Glycosylation; D006801:Humans; D010316:Particle Size; D010442:Peptide Chain Initiation, Translational; D010957:Plasmids; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D050436:Regulatory Elements, Transcriptional; D013205:Staphylococcal Protein A / Q000737:chemistry; D014162:Transfection
D000911:Antibodies, Monoclonal; D011134:Polysaccharides; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A