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KMID : 1094720130180020419
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Biotechnology and Bioprocess Engineering
2013 Volume.18 No. 2 p.419 ~ p.429
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Analysis of heterogeneity and instability of stable mAb-expressing CHO cells
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Du Zhimei
Mujacic Mirna
Le Kim
Caspary Guy
Nunn Heather
Heath Carole
Reddy Pranhitha
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Abstract
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Screening and isolation of high expression mammalian cell lines for production of recombinant proteins for the clinic is a resource-intensive and time-consuming procedure due to the substantial variation in expression levels of recombinant protein expression amongst transfected cells. Several investigators have reported instability in expression titers early in cell line development and in cell banks. However, in most cases the exact molecular mechanisms of instability remain unknown. In this study we used a fluorescence-activated cell sorting (FACS) based mAb staining method to enable the detection and selective gating of cells with vastly different recombinant expression levels present in transfected pools. Expression diversity and changes within transfected populations were detected and isolated in real time during cell line development. Molecular genetic analysis on the isolated clones revealed an unsuspected rearrangement of the heavy chain in the non-expressing clones. Implications of the genetic rearrangements as well as the use of the FACS method as a tool to improve cell line development to detect expression heterogeneity in pools and to investigate root cause for the molecular genetics of expression instability will be discussed.
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KEYWORD
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FACS, recombinant antibody, production, CHO, cell line development
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