Abstract
BackgroundThe establishment of high producer is an important issue in Chinese hamster ovary (CHO) cell culture considering increased heterogeneity by the random integration of a transfected foreign gene and the altered position of the integrated gene. Fluorescence-activated cell sorting (FACS)-based cell line development is an efficient strategy for the selection of CHO cells in high therapeutic protein production.ResultsAn internal ribosome entry site (IRES) was introduced for using two green fluorescence protein (GFP) fragments as a reporter to both antibody chains, the heavy chain and the light chain. The cells co-transfected with two GFP fragments showed the emission of green fluorescence by the reconstitution of split GFP. The FACS-sorted pool with GFP expression had a higher specific antibody productivity (qAb) than that of the unsorted pool. The qAb was highly correlated with the fluorescence intensity with a high correlation coefficient, evidenced from the analysis of median GFP and qAb in individual selected clones.ConclusionsThis study proved that the fragment complementation for split GFP could be an efficient indication for antibody production on the basis of high correlation of qAb with reconstitution of GFP. Taken together, we developed an efficient FACS-based screening method for high antibody-producing CHO cells with the benefits of the split GFP system.
Highlights
The establishment of high producer is an important issue in Chinese hamster ovary (CHO) cell culture considering increased heterogeneity by the random integration of a transfected foreign gene and the altered position of the integrated gene
We developed a new cell screening method for high antibody-producing CHO cells based on the reassembly of split green fluorescence protein (GFP) combined with Fluorescence-activated cell sorting (FACS)
We found that the FACS-sorted cells with GFP expression are the antibody-producing CHO cells
Summary
The establishment of high producer is an important issue in Chinese hamster ovary (CHO) cell culture considering increased heterogeneity by the random integration of a transfected foreign gene and the altered position of the integrated gene. Fluorescence-activated cell sorting (FACS)-based cell line development is an efficient strategy for the selection of CHO cells in high therapeutic protein production. Chinese hamster ovary (CHO) cells are one of the most widely used host cells for therapeutic protein production. The selection of CHO cells for high therapeutic protein production is of particular interest in CHO cell culture for a wide variety of individual clones for productivity from a random integration and gene amplification system [1]. Due to the large number of candidates that need their productivity evaluated, an efficient highthroughput cell screening system needs to be developed. Flow cytometry can efficiently evaluate a number of cells at the single-cell level in a short time and isolate a single clone from sub-populations. Various fluorescenceactivated cell sorting (FACS)-based cell screening methods have been developed for CHO cells with the benefits of flow cytometry [1]. The use of an antibody conjugated protein by internal ribosome entry site (IRES)-linking and the high producer is detected using a fluorescent-conjugated anti-CD20 antibody [5]
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