Characterisation of recombinant CHO cell lines by investigation of protein productivities and genetic parameters.
We have generated a recombinant CHO cell line expressing the fusion protein EpoFc. After selection and screening, protein expression, gene and mRNA copy numbers were analysed in order to gain more information on the influence of genetic parameters on the productivity and stability of production cells. Results from semi-quantitative blot methods were compared to quantitative PCR (qPCR) analyses, whose advantage mainly lies in their higher sensitivity, and the cheaper and faster methodology. We developed stable and high producing clones with low gene copy numbers, in contrast to other cell lines where multiple steps of methotrexate amplification have lead to hundreds of copies of inserts with the risk of karyotypic instabilities and decreased growth rates that overcome the benefits of increased productivities. When comparing genetic parameters to productivity, a good correlation of mRNA levels with specific productivity was observed, whereas high gene copy numbers were not always accompanied by high protein expressions. Based on our data derived from a typical example of a cell line development process, genetic parameters are useful tools for the selection of scalable production clones. Nevertheless, a wider range of cell lines has to be investigated in order to implement genetic analyses into a screening process.
Lattenmayer
Christine
C
Austrian Center of Biopharmaceutical Technology, Muthgasse 18, 1190 Vienna, Austria. christine.lattenmayer@malteser.at
Trummer
Evelyn
E
Schriebl
Kornelia
K
Vorauer-Uhl
Karola
K
Mueller
Dethardt
D
Katinger
Hermann
H
Kunert
Renate
R
D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D016466:CHO Cells; D015815:Cell Adhesion Molecules / Q000096:biosynthesis* / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D004921:Erythropoietin / Q000096:biosynthesis* / Q000235:genetics; D018628:Gene Dosage; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics
D015815:Cell Adhesion Molecules; C106607:FCGBP protein, human; D008565:Membrane Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D004921:Erythropoietin