Advances in Clone Selection Using High-Throughput Bioreactors Bhargavi Kondragunta Center for Advanced Sensor Technology, University of Maryland, Baltimore County, Baltimore, MD 21250 Dept. of Chemical and Biochemical Engineering, University of Maryland, Baltimore County, Baltimore, MD 21250 Div. of Monoclonal Antibodies, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20903 Jessica L. Drew Center for Advanced Sensor Technology, University of Maryland, Baltimore County, Baltimore, MD 21250 Dept. of Chemical and Biochemical Engineering, University of Maryland, Baltimore County, Baltimore, MD 21250 Kurt A. Brorson Div. of Monoclonal Antibodies, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20903 Antonio R. Moreira and Govind Rao Center for Advanced Sensor Technology, University of Maryland, Baltimore County, Baltimore, MD 21250 Dept. of Chemical and Biochemical Engineering, University of Maryland, Baltimore County, Baltimore, MD 21250 DOI 10.1002/btpr.392 Published online January 29, 2010 in Wiley Online Library (wileyonlinelibrary.com). Effective clone selection is a crucial step toward developing a robust mammalian cell culture production platform. Currently, clone selection is done by culturing cells in well plates and picking the highest producers. Ideally, clone selection should be done in a stirred tank bioreactor as this would best replicate the eventual production environment. The actual number of clones selected for future evaluation in bioreactors at bench-scale is limited by the scale-up and operational costs involved. This study describes the application of miniatur- ized stirred high-throughput bioreactors (35 mL working volume; HTBRs) with noninvasive optical sensors for clone screening and selection. We investigated a method for testing several subclones simultaneously in a stirred environment using our high throughput bioreactors (up to 12 clones per HTBR run) and compared it with a traditional well plate selection approach. Importantly, it was found that selecting clones solely based on results from stationary well plate cultures could result in the chance of missing higher producing clones. Our approach suggests that choosing a clone after analyzing its performance in a stirred bioreactor environment is an improved method for clone selection. V V C 2010 American Institute of Chemical Engineers Biotechnol. Prog., 26: 1095–1103, 2010 Keywords: high-throughput, mini-bioreactors, subcloning, noninvasive, optical, sensors Introduction Therapeutic proteins have an annual global market of US $33 billion, and it is expected to increase to US $70 billion by 2010. 1 The success of engineered therapeutic monoclonal antibodies in particular has spurred considerable interest in developing improved manufacturing strategies, including initial selection of high-producing stable antibody expressing clones in a cost-effective and high-throughput manner. Effective subclone selection, typically done after transfection, is central to obtaining stable cell lines for man- ufacturing. In addition, subcloning of hybridomas must also be done periodically to remove low and nonproducing cells that arise as a result of chromosome loss and mutation in the heavy chain of the antibody. 2 In many cases, nonproducers have been found to proliferate faster than those cells that are producing higher antibody titers 3 and overtake the culture. The traditional method of subclone selection is by limiting dilution (LD) cloning in 96-well plates, generally the most economical and simplest method. 4 The highest producing clone is chosen based on ELISA results from the stationary well plates. In some cases, cells are selected based on spe- cific productivity of membrane IgG expression of individual cells by cell sorting. 5 Data from other groups has indicated that the selection methods based on productivity from a sin- gle point resulted in a high chance of selecting an unstable cell line. 6 Clone selection should be based not only on higher titers in 96-well cultures, but also on compatibility with the actual production culture such as batch or fed-batch processes. A major drawback with the 96-well plate approach is time and expense; developing and qualifying such stable cell lines with proven higher antibody productiv- ity is a major undertaking in a commercial environment that Statements in this article represent the views of the authors and do not constitute official positions or policies of the Food and Drug Admin- istration or the U.S. Government. Correspondence concerning this article should be addressed to Govind Rao at grao@umbc.edu V V C 2010 American Institute of Chemical Engineers 1095