Accelerated clone selection for recombinant CHO CELLS using a FACS-based high-throughput screen.
Flow cytometry was partnered with a nonfluorescent reporter protein for rapid, early stage identification of clones producing high levels of a therapeutic protein. A cell surface protein, not normally expressed on CHO cells, is coexpressed, as a reporter, with the therapeutic protein and detected using a fluorescently labeled antibody. The genes encoding the reporter protein and the therapeutic protein are linked by an IRES, so that they are transcribed in the same mRNA but are translated independently. Since they each arise from a common mRNA, the reporter protein's expression level accurately predicts the relative expression level of the therapeutic protein for each clone. This method provides an effective process for generating recombinant cell lines producing high levels of therapeutic proteins, with the benefits of rapid and accurate 96-well plate clone screening and elimination of unstable clones at an earlier stage in the development process. Furthermore, because this method does not rely on the availability of an antibody specific for the therapeutic protein being expressed, it can be easily implemented into any cell line development process.
DeMaria
Christine T
CT
Therapeutic Protein Expression Group, Department of Cell & Protein Therapeutics, Genzyme Corporation, Framingham, Massachusetts 01701, USA. christine.demaria@genzyme.com
Cairns
Victor
V
Schwarz
Cordula
C
Zhang
Jin
J
Guerin
Megan
M
Zuena
Erin
E
Estes
Scott
S
Karey
Kenneth P
KP
D000818:Animals; D018951:Antigens, CD20 / Q000032:analysis*; D016466:CHO Cells / Q000145:classification / Q000166:cytology* / Q000276:immunology*; D002469:Cell Separation / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry / Q000379:methods*; D011995:Recombination, Genetic / Q000502:physiology*
D018951:Antigens, CD20