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 to quantify organelle

content, and recombinant heavy (HC) and light (LC) chain polypeptide and mRNA amounts in

single cells were developed. The assays were then validated against orthogonal standard flow

cytometry, western blot and qRT-PCR methods. We 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. Our analysis found HC polypeptide and mRNA to be predictive of productivity early

in the culture, however specific organelle content did not show any correlation with

productivity.