Accelerating biologics discovery and cell development with picodroplets technology

This article is based on a poster originally authored by Maryam Ahmadi, Elena Shvets, Frank Gesellchen, Xin Liu, Jessica Hall and Richard Hammond, which was presented at ELRIG Drug Discovery 2024 in affiliation with Sphere Fluidics Limited.

This poster is being hosted on this website in its raw form, without modifications. It has not undergone peer review but has been reviewed to meet AZoNetwork's editorial quality standards. The information contained is for informational purposes only and should not be considered validated by independent peer assessment.

Although antibody therapeutics dominate drug development, prolonged workflows and increasing costs hinder their success during their discovery and development.^1-3 Cyto-Mine^® offers significant advantages in the number of cells that can be processed, cost per run, and the speed with which projects can be completed.

Sphere Fluidics has developed Cyto-Mine^® Chroma, a new instrument that enables the use of more fluors, multiplexed assays, and a broader range of applications.

Early applications of the Cyto-Mine^® Chroma platform will enable cell isolation based on cell surface markers, viability, and titer of secreted proteins. Re-injection-based workflows facilitate complex assays like combining more than one cell type in a single droplet (crucial for binding assessment, cytotoxic assays, and advancing antibody development).

Cyto-Mine^®

Figure 1. The Cyto-Mine^® platform. Image Credit: Maryam Ahmadi et al., in partnership with Sphere Fluidics.

Figure 2. A Cyto-Mine^® workflow. Integration of single-cell screening, sorting, isolation, and verification using a fully integrated microfluidic process. Image Credit: Maryam Ahmadi et al., in partnership with Sphere Fluidics.

Table 1. Features of Cyto-Mine^® and Cyto-Mine^® Chroma. Source: Sphere Fluidics

Cyto-Mine^® harnesses picodroplet technology to find rare and valuable variants within heterogeneous cell samples. The machine integrates single-cell encapsulation (generation), a fluorescence-based optical detection method followed by sorting for isolation of ‘hits,’ verification by imaging of monoclonality assurance, and single-cell dispensing into microtiter plates.

Increasing efficiency of cell line development processes with Cyto-Mine^®

Cyto-Mine^® increases the success rate in gene integration, clone selection, and monoclonality assurance

Figure 3. Traditional CLD processes are time-consuming. Cyto-Mine^® reduces time and increases success rates. At a single-cell level, the clones are selected based on their productivity. Selected single cells are dispensed into microplates with image assurance on their monoclonality. Cells dispensed into microplates can be monitored for growth over time. Image Credit: Maryam Ahmadi et al., in partnership with Sphere Fluidics.

Gene integration - increasing efficiency using picodroplets

Figure 4. A) Compatibility of picodroplets with different transfection reagents. B) Cells transfected with DNA (GFP)/Lipofectamine 3000 in picodroplets C) 4 x increase in transfection efficiency in picodroplets vs. control. Image Credit: Maryam Ahmadi et al., in partnership with Sphere Fluidics.

Clone selection using FRET assays

Figure 5. Clone Selection using CytoCellect^®PLUS. A&B) The kit detects secreted IgG antibodies regardless of their light chain. C) The kit can be used with Cyto-Mine^® to select high-producing clones, even if the IgG light chain (kappa or lambda) is unknown. Image Credit: Maryam Ahmadi et al., in partnership with Sphere Fluidics.

Clone selection - using FC fusion to determine productivity

Figure 6. Clone selection for cells secreting Fc Fusion proteins. A) A quantitative competition FRET assay used to detect human Fc fusion proteins. B) Can be used to detect Fc fusion proteins in a plate reader assay format. C) Can also be used with Cyto-Mine^® to detect secreted protein. Image Credit: Maryam Ahmadi et al., in partnership with Sphere Fluidics.

Monoclonality assurance

• Cyto-Mine^® has been specifically designed to ensure monoclonality via accurate and reliable single-cell dispensing.
• Cyto-Mine^® dispenses and reports clonality of cells with an accuracy of 98.9 %.

Figure 7. A) Cyto-Mine^® visual verification and dispensing. Immediately before dispensing, picodroplets are brightfield-imaged to identify and record the number of cells per picodroplet and re-measured for picodroplet fluorescence. B) Brightfield multi-frame imaging avoids potential miscounting by detecting encapsulated cells at multiple timepoints as they rotate into different positions. Image Credit: Maryam Ahmadi et al., in partnership with Sphere Fluidics.

Growth and stability - Maintaining outgrowth

Figure 8. Cell outgrowth using Cyto-Mine^® vs Limited Dilution Cloning (LDC). Image Credit: Maryam Ahmadi et al., in partnership with Sphere Fluidics.

• Single cells isolated using Cyto-Mine^® have high viability and show comparable growth compared to Limited Dilution Cloning

Enabling antibody discovery using Cyto-Mine^®

Cyto-Mine^® brings speed and simplicity to antibody discovery processes

Figure 9. Antibody discovery workflow using Cyto-Mine^®. Image Credit: Maryam Ahmadi et al., in partnership with Sphere Fluidics.

B cell repertoire screening

Figure 10. High-throughput single B cell screening using whole B cell repertoire. B cells are selected based on IgG and/or antigen specificity secretion. Image Credit: Maryam Ahmadi et al., in partnership with Sphere Fluidics.

• Cyto-Mine^® is used for high-throughput B cell screening and isolating rare cells based on their productivity.

Figure 11. High sensitivity isolation of rare single cells based on their antigen specificity. Image Credit: Maryam Ahmadi et al., in partnership with Sphere Fluidics.

Developing Cyto-Mine^® Chroma

Going beyond gene integration

Figure 12. Multiplexing enables the isolation of clones based on the efficiency of DNA integration, viability, and titer. Image Credit: Maryam Ahmadi et al., in partnership with Sphere Fluidics.

• Transfection takes place in picodroplets
• Cells are incubated for up to 72 hours, and the most productive, viable cells are isolated using Cyto- Mine^® Chroma

A more efficient selection process

Figure 13. Example selection process. Hybridoma A: Labelled with CellTracker Blue; produces irrelevant IgG. Hybridoma B: Labelled with CellTracker Deep Red, produces an anti-TNF-alpha antibody. Image Credit: Maryam Ahmadi et al., in partnership with Sphere Fluidics.

Figure 14. Single cells producing anti-TNF-alpha were detected and isolated using Cyto-Mine^® Chroma (recombinant TNF-alpha was used for antigen-specific FRET assay). Image Credit: Maryam Ahmadi et al., in partnership with Sphere Fluidics.

• Multiplexing assays in a single droplet improves the selection process of high-producing clones
• Cells can be imaged for viability, productivity, and antigen specificity in a single step
• Additional applications include mode of action assays involving multiple cells per droplet, where the function of a labeled cell type is evaluated in the presence of an unlabeled cell within picodroplets.

Expanding Cyto-Mine^® applications: Re-injection

1. Off-line generated picodroplets are injected into Cyto-Mine^®
2. Droplets are sorted and dispensed using Cyto-Mine^®

Relevant applications include:

• Applications requiring long incubation of cells in droplets
• Applications requiring extensive manipulation during droplet generation

Figure 15. Off-line generated droplets are stable for prolonged periods of time and can be re-injected into Cyto-Mine^®. Droplets were generated and incubated at 37 °C, 5 % CO₂ for up to 48 h. (A) Microscopy images were taken at 0, 24, and 48-hour time points. (B) In the presence of FRET probes, droplets containing mouse IgG (4 different concentrations) were re-injected into Cyto-Mine^®. Data shows that prolonged incubation does not affect droplet stability. Image Credit: Maryam Ahmadi et al., in partnership with Sphere Fluidics.

References:

1. Kaplon, H., et al. (2022). Antibodies to watch in 2022. mAbs, 14(1). https://doi.org/10.1080/19420862.2021.2014296.
2. Bauer, J., et al. (2023). How can we discover developable antibody-based biotherapeutics? Frontiers in Molecular Biosciences, [online] 10, p.1221626. https://doi.org/10.3389/fmolb.2023.1221626.
3. Farid, S.S., et al. (2020). Benchmarking biopharmaceutical process development and manufacturing cost contributions to R&D. mAbs, [online] 12(1), p.1754999. https://doi.org/10.1080/19420862.2020.1754999.

About Sphere Fluidics

Our vision

Our philosophy is simple. We combine our knowledge and resources to help you find rare and valuable biological variants, while helping you to save time, reduce costs and stay a step ahead of the competition.

Our novel single cell analysis systems offer the rapid screening and characterization of single cells. These systems are underpinned by our patented picodroplet technology, specifically designed to increase your chances of finding that rare ‘one-in-a-billion’ molecule or cell that could be an industry blockbuster.

We understand that time is of the essence. That’s why our technologies boost throughput and assay sensitivity across a range of applications. Most importantly, our flexible systems evolve alongside your changing research needs, providing an adaptable platform that helps you to meet your goals.

Our history

Founded in 2010, Sphere Fluidics is an established Life Sciences company, originally spun out from the University of Cambridge. We initially developed 25 patented products – biochips and specialist chemicals – which currently assist hundreds of customers globally with their research.

We initially focused on producing novel biochip systems and providing R&D services. We have since extended our expertise and are developing a technology platform that enables discovery in a range of growing markets through single cell analysis. Our systems make the development of new biopharmaceuticals faster and more cost-effective, improve monoclonal antibody screening, cell line development, and overall research efficiency in a number of other applications including synthetic biology, single cell diagnostics, prognostics and single cell genome editing.

The Cyto-Mine^® Single Cell Analysis System is our flagship product – the first integrated, benchtop system to automatically analyse, sort and dispense millions of individual cells in just a single day.

Our partnerships

We value and are always open to discussing new collaborative, successful and innovative academic and industry partnerships to further develop and improve our single cell technologies.

Our Technology Access Programmes and Collaborative Services exist to enable academic researchers and companies alike to tap into our application-specific expertise through direct partnerships.

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