Application of Imaging Flow Cytometry for the Characterization of Intracellular Attributes in Chinese Hamster Ovary Cell Lines at the Single-Cell Level.

Biopharmaceutical manufacturing using Chinese hamster ovary (CHO) cells requires the generation of high-producing clonal cell lines. During cell line development, cell cloning using fluorescence-activated cell sorting (FACS) has the potential to combine isolation of single cells with sorting based on specific cellular attributes that correlate with productivity and/or growth, identifying cell lines with desirable phenotypes for manufacturing. This study describes the application of imaging flow cytometry (IFC) to characterize recombinant cell lines at the single-cell level to identify cell attributes predictive of productivity. IFC assays are developed to quantify the organelle content and recombinant heavy-chain (HC) and light-chain (LC) polypeptide as well as messenger RNA (mRNA) amounts in single cells. The assays are then validated against orthogonal standard flow cytometry, western blot, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) methods. The authors describe how these IFC assays may be used in cell line development and show how cellular properties can be correlated with productivity at the single-cell level, allowing the isolation of such cells during the cloning process. From the analysis, HC polypeptide and mRNA are found to be predictive of productivity early in the culture; however, specific organelle content did not show any correlation with productivity.