Split-GFP and Droplet Microfluidics for High-Throughput Screening of Mammalian Cell Factories

The industrial production of monoclonal antibodies and other protein therapeutics is currently mainly achieved by heterologous overexpression of recombinant genes in Chinese hamster ovary (CHO) cells. These cells are popular for a multitude of reasons, in particular for their ability to generate folded and glycosylated proteins at high yield at large scale. To meet the growing demands of high-yielding and cost-effective production strains numerous rational, undirected and combinatorial approaches have been employed to enhance the level of protein secretion. The lack of access to scalable and fast ways of monitoring product secretion is however limiting the ability for a complete and efficient screening of such large libraries of cells.

Here we present a high throughput screening system allowing for single cell monitoring of protein secretion and fluorescent sorting using a combination of droplet microfluidics and split-GFP complementation. Briefly a short tag, comprising the 11-strand of GFP, was genetically fused to either the monoclonal antibody Herceptin or the glycoprotein EPO and expressed in CHO as fusion proteins. The expressed fusion protein will remain non-fluorescent until complemented with the remaining part of the GFP protein (GFP 1-10). We demonstrate the ability to encapsulate and cultivate single CHO cells in mono-disperse droplets containing cultivation medium supplemented with GFP 1-10 and that we are able to monitor protein secretion of single cells by fluorescence and to sort CHO cell factory libraries based on their protein secretion level. Further we show the ability to de-tag the 11^th strand from the high producing clones once identified using Crispr/Cas9. We believe this generic technology will allow for a better ability to perform thorough and fast screenings of CHO cell factories for the identification of high-secreting production clones.