Antibody-derived biologics have become a major class of modern medicine, particularly in the fight against cancer and autoimmune diseases. Crucial to the successful translation of antibodies into therapies are highly efficient antibody discovery and cell line development pipelines. The challenge for biopharma is to screen large cell populations for productivity, antigen specificity, or other parameters, and then isolate rare cells with assured clonality.

Traditionally, this was achieved by the resource-intensive method of limiting dilution, and more recently aided by semiautomated technologies such as fluorescence-activated cell sorting (FACS), colony picking, and cell-in-well imagers, but these offer only partial solutions for automated workflows. High-throughput cell-sorting methods cannot readily measure secreted protein, and current secreted protein screening methods are limited in their ability to handle large numbers of cells. As a result, the screening, cloning, and verification steps for antibody discovery and cell line development still require manual intervention at each stage, creating substantial bottlenecks.

Next-Generation Single-Cell Analysis

Picodroplet cell encapsulation technology is a unique approach to single-cell analysis that can deliver higher throughputs, rapid yet gentle cell processing, single-cell dispensing to microplates, and monoclonality verification by imaging, in a fully automated platform.

Sphere Fluidics’ Cyto-Mine® Single Cell Analysis and Monoclonality Assurance System is a next-generation single-cell analysis platform that integrates antibody discovery processes using patented picodroplet technology to encapsulate single cells, which can then be assayed and sorted automatically (Figure 1).

Sphere Fluidics Figure 1
Figure 1. Schematic of the automated Cyto-Mine® workflow, which integrates cell screening, sorting, and monoclonality assurance by imaging into a single platform using picodroplet technology.


1) Cells encapsulated into picodroplets: The target cell population is prepared in preferred culture medium supplemented with an appropriate animal-origin-free (AOF) antibody-based detection reagent for the selected secretion assay. The cell suspension is then gently pumped through microfluidic channels and mixed with a biocompatible surfactant, which encapsulates a single cell (or pools of cells) in each 300-pL droplet of culture medium.

2) Incubation and secreted protein assay: Approximately two million generated picodroplets are collected together into a chamber and incubated in situ at 37°C to activate the required assay. The assay could be used to detect secreted proteins, biomarkers,  intracellular fluorescence (such as a viability stain), or an intrinsic functional assay marker.

3) Sorting positive cells: The picodroplets are sorted by fluorescence detection and gating, similar to typical flow cytometry, with positives being actively channeled for collection. The population of cells selected for collection can be defined and adjusted according to each specific experiment. Selected positive picodroplets are stored in a chilled microchamber prior to dispensing, and negative picodroplets that contain low-fluorescing cells, or are empty, are diverted to waste.

4) Visual verification and dispensing: After completion of the sorting phase, positive picodroplets are selected, imaged, and dispensed to individual wells of a 96- or 384-well microplate prefilled with preferred culture medium. The imaging process uses an ultra-high-speed brightfield camera to acquire multiple frames of each picodroplet, providing unambiguous visual evidence of a single cell progenitor.

FRET-Based Protein Secretion Assays

Förster resonance energy transfer (FRET)-based assays are used in the Cyto-Mine platform to detect protein(s) secreted by encapsulated cells. The assay can be customized by designing AOF fluorescent detection probes specific for the protein of interest, such as IgG (e.g., for a productivity screen) or antigen-specific IgG (e.g., for a hybridoma fusion screen or B-cell mining). When the secreted protein is recognized by the detection probes, a three-body FRET complex is formed, which induces a FRET-mediated shift in fluorescence. This signal is used to determine the quantity of secreted protein and select picodroplets for collection.

IgG Secretion Assay for Cell Line Development

Sphere Fluidics developed an assay to measure the IgG production rate of single cells in a sample population, using a customized pair of IgG-specific fluorescent probes (Figure 2A). To validate the assay, five populations of picodroplets generated from medium containing varying concentrations of IgG were pooled together, detected by Cyto-Mine and analyzed using Cyto-Mine Studio Software. Five discrete picodroplet populations could be resolved, corresponding to the different IgG concentrations, confirming that the assay could quantitate IgG secretion (Figures 2B & 2C).

Sphere Fluidics Figure 2
Figure 2. Cyto-Mine IgG secretion assay. (A) IgG-specific probes are trapped within each picodroplet and bind to secreted IgG to form a three-body FRET complex. (B) Scatterplots of FRET signal for picodroplets containing the indicated concentrations of human IgG with IgG detection probes. (C) Standard titration curve derived from data in B. (D) Scatterplot generated from picodroplet-encapsulated CHO cells incubated with IgG-specific detection probes.

The IgG assay was then used to screen a heterogeneous pool of Chinese hamster ovary (CHO) cells, stably transfected to express human IgG, to identify and isolate the highest antibody-producing clones. The population of picodroplets to the upper left of the scatterplot (Figure 2D) with high acceptor-to-donor fluorescence ratio was gated for collection. The bright cluster to the lower right represents the bulk of data points, which comprises picodroplets containing low- or nonproducing cells and empty picodroplets.

Antigen-Specificity Assay for Antibody Discovery

Sphere Fluidics also carried out a study to screen hybridomas for antigen-specific clones generated from a mouse immunized with human tumor necrosis factor-α (TNF-α). The detection probes comprised fluorescently conjugated human TNF-α and a mouse IgG-Fc acceptor probe, enabling specific detection of only mouse IgG-recognizing human TNF-α (Figure 3A). The choice of probes permits simultaneous screening for antigen-specificity and immunoglobulin isotype.

Sphere Fluidics Figure 3
Figure 3. Cyto-Mine antigen-specificity assay. (A) Antigen-specific IgG secreted from the encapsulated cell is recognized by the fluorescently conjugated antigen and the IgG-specific probe. (B) Scatterplots of FRET signal from picodroplets with the indicated concentrations of anti-human TNF-α IgG, detected by fluorescently conjugated human TNF-α and an IgG-specific acceptor probe. (C) Representative titration curve generated from data in B. (D) Scatterplot of FRET signal generated from hybridomas screened for secretion of anti-human TNF-α IgG.

Titration experiments were undertaken with culture medium picodroplets containing different concentrations of anti-human TNF-α IgG. Different titers could be resolved into discrete picodroplet populations, and the control IgG experiment demonstrated the fidelity of the assay for detecting only antigen-specific IgG (Figures 3B & 3C).

A mixed population of hybridoma cells was analyzed with the validated detection probes to find TNF-α-specific IgG-producing clones. A subpopulation of cells with a high acceptor-to-donor fluorescence ratio, indicating secretion of human TNF-α-specific IgG, was gated for collection, while the majority of picodroplets were diverted to waste (Figure 3D).

Beyond Antibody Discovery

Biopharmaceutical organizations are under increasing pressure to streamline their antibody discovery and cell line development processes, with unmet needs for increased throughput, shortened timelines, reduced costs, and improved proof of monoclonality. Next-generation, single-cell analysis platforms like the Cyto-Mine allow researchers to screen hundreds of thousands of individual cells or up to 40 million cells (in pools) for secreted target protein, to isolate high-value cells or pools of interest, and to dispense with high viability into microplate wells—and to accomplish these tasks more efficiently by using one integrated and automated system. The system can be tailored to a range of requirements and workflows in bioproduction and other biological areas, such as genome editing, single-cell disease diagnostics, and metagenomics. All could benefit from the improved efficiencies that are achieved by harnessing picodroplet technology.


Frank F. Craig, Ph.D. ([email protected]), is CEO of Sphere Fluidics.

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