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As the COVID-19 pandemic revealed, rapid development of vaccines and therapeutic antibodies are crucial to guarantee a quick return to the status quo of society. In early 2020, we deployed our droplet microfluidic single-cell-based platform DROPZYLLA ® for the generation of cognate antibody repertoires of convalescent COVID-19 donors. Discovery of SARS-CoV-2-specific antibodies was performed upon display of antibodies on the surface of HEK293T cells by antigen-specific sorting using binding to the SARS-CoV-2 spike and absence of binding to huACE2 as the sort criteria. This efficiently yielded antibodies within 3-6 weeks, of which up to 100% were neutralizing. One of these, MTX-COVAB, displaying low picomolar neutralization IC50 of SARS-CoV-2 and with a neutralization potency on par with the Regeneron antibodies, was selected for GMP manufacturing and clinical development in June 2020. MTX-COVAB showed strong efficacy in vivo and neutralized all identified clinically relevant variants of SARS-CoV-2 at the time of its selection. MTX-COVAB completed GMP manufacturing by the end of 2020, but clinical development was stopped when the Omicron variant emerged, a variant that proved to be detrimental to all monoclonal antibodies already approved. The present study describes the capabilities of the DROPZYLLA ® platform to identify antibodies of high virus-neutralizing capacity rapidly and directly.

High-throughput screening and enrichment of antibody-producing cells have many important applications. Herein, we present a droplet microfluidic approach for high-throughput screening and sorting of antibody-secreting cells using a Förster resonance electron transfer (FRET)-based assay. The FRET signal is mediated by the specific binding of the secreted antibody to two fluorescently labeled probes supplied within a droplet. Functional hybridoma cells expressing either membrane-bound or secreted monoclonal antibodies (mAbs), or both, were efficiently differentiated in less than 30 min. The antibody secretion rate by individual hybridoma cells was recorded in the range of 14,000 Abs/min, while the density of membrane-bound fraction was approximately 100 Abs/μm 2 . Combining the FRET assay with droplet-based single-cell sorting, an 800-fold enrichment of antigen-specific cells was achieved after one round of sorting. The presented system overcomes several key limitations observed in conventional FACS-based screening methods and should be applicable to assaying various other secreted proteins., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)