Efficient selection of high-producing subclones during gene amplification of recombinant Chinese hamster ovary cells by flow cytometry and cell sorting

bThe screening procedure for high-producing cell lines is extremely time- a nd labor-intensive and costly, and is at present guided by an empirical app roach based on individual experience. Flow cytometry and cell sorting, with its ability to analyze and separate single cells, an ideal method in the s election of such rare cells. The isolation of recombinant cell lines is esp ecially difficult due to repeated gene amplification, which introduces high mutational variation into the population. We have established and evaluate d a modification of a previous method that traps secreted product on the su rface of the secreting cell, thus allowing direct analysis of single cell s pecific production rates. This method was used to select for high-producing subclones of a recombinant Chinese hamster ovary (CHO) cell line producing a human antibody against HIV-1 by repeated rounds of gene amplification an d cell sorting. This cell line has been amplified in previous investigation s, so that the amount of work and testing required by traditional methods c an be compared with the protocol described herein. Forty-five 96-well plate s were necessary to obtain a high-producing subclone by limited dilution me thods, whereas only five plates were required when cell sorting was used. T he specific production rate of the best clone obtained by sorting, however, was five times that of the clone obtained by traditional methods. In contr ast to the clones obtained by limited dilution, which consisted of several populations of low- and high-producing cells even at high methotrexate conc entrations (6.4 muM), the clones isolated by sorting were already homogeneo us at 0.8 muM methotrexate. (C) 2001 John Wiley & Sons, Inc.