The increase in the utilization of monoclonal antibodies as therapeutics over the past two decades has been remarkable, with over 80 mAbs approved for use.
The quick pace of clinical studies and approvals in the antibody-based therapeutics sector is generating a significant demand for biopharmaceutical companies to shorten the time it takes to bring new biologics to market.
Traditional methods for screening large cell panels used in antibody discovery during lead generation are demanding in terms of resources and labor, requiring multiple pieces of equipment for each step, such as single-cell analysis, sorting, imaging, and dispensing into individual wells of microtiter plates, resulting in major bottlenecks in identifying lead candidates.
Cyto-Mine® helps to address the time-consuming, labor-intensive nature of screening large cell populations for rare antigen-specific, antibody-secreting cells during antibody discovery by streamlining the traditional multi-step process into one seamless, fully-integrated one-day workflow.
Hybridoma-based antibody discovery
The discovery of antibodies often begins with the identification of a known, validated target. The next step is to generate candidate antibodies for hit selection.
Figure 1. Traditional workflow in hybridoma screening. Image Credit: Sphere Fluidics
Historically, immunization of animals, such as mice, rats, or rabbits, is used to induce an immune response. After several weeks, B-cells can be harvested and merged with myeloma cells to form hybridoma populations (Figure 1).
An alternative approach involves directly isolating B-cells. Regardless of the method used, extensive screening and characterization of the cells producing the antibodies are necessary. However, conventional screening methods, such as flow cytometry and ELISA, can result in bottlenecks.
While flow cytometry is a high-throughput method for screening large cell populations, it has several limitations.
The harsh process of flow cytometry can cause shear stress that can damage cells, including rare or valuable ones, thereby decreasing their viability and potential for recovery.
Additionally, flow cytometry is not an effective method for measuring cell secretion. Cold capture secretion assays can retain secreted molecules at the cell surface through cell manipulation, but any uncaptured molecules will spread into the extracellular environment, resulting in an inaccurate representation of cell secretion.
Once the antibodies have been screened and characterized, they must be subcloned into monoclonal populations.
This process involves the use of semi-automated technologies, such as cell printers and cell-in-well imagers, which increase the number of instruments involved in the workflow, making it more time-consuming and labor-intensive.
Figure 2. Accelerated workflow in hybridoma screening. Image Credit: Sphere Fluidics
Cyto-Mine® is a fully integrated platform that uses microfluidic picodroplet technology to streamline the discovery and development of antibody-based therapeutics.
By combining cell isolation, assay, sorting, imaging, and dispensing into a single automated workflow, Cyto-Mine® reduces the timeline for antibody discovery (as shown in Figure 2).
Image Credit: Sphere Fluidics
The platform addresses the major challenges in the antibody discovery workflow, including:
- Flexibility: Adaptable assay design for specific needs
- Measurement: Semi-quantitative assays of antibody secretion
- Sensitivity and Specificity: Detect antibodies of interest
- Efficiency: Screen the entire cell population
- Viability: Maintain high levels of cell viability
- Speed: Reduce total workflow timelines
Picodroplet. Image Credit: Sphere Fluidics
Sphere Fluidics provides antigen-specific assays for the detection of specific targets. The company offers flexible assay design, including the capability to create custom-made assays to meet specific application requirements. These assays can include antigen-specific assays for hybridoma screening or B-cell mining.
Picodroplet-based technologies offer a highly sensitive way to assay and select antigen-specific, antibody-secreting cells that conventional methods cannot.
To demonstrate the semi-quantitative nature of the Cyto-Mine® antigen-specific assay, experiments were performed using human TNF-alpha as a model antigen. The results, shown in Figure 3, illustrate how different concentrations of the antigen were resolved into distinct populations.
As a control, the samples were analyzed along with control IgG that did not express the antigen using a fluorescent plate reader (Figure 4).
The titration curve in Figure 4 demonstrates that the antigen-specific assay probes only detected the human TNF-alpha population and not the control IgG, indicating that the assay is specific to the antigen of interest.
Finally, in Figure 5, a hybridoma population secreting human TNF-alpha-specific IgG at unknown concentrations was analyzed to show the spread of production capacities among the hybridoma population and to demonstrate that the assay reagents could identify secreted antigen-specific antibodies.
Figure 3. Cyto-Mine® Scatter Plot. A library of picodroplets was made containing different concentrations of target anti-human TNF-alpha IgG and detected using an antigen-specific assay. Image Credit: Sphere Fluidics
Figure 4. Representative Titration Curve. Generated using the Cyto-Mine® antigen-specific assay. In this example, the control IgG confirms specificity of the assay for human TNF-alpha. Image Credit: Sphere Fluidics
Figure 5. Cyto-Mine® Scatter Plot. FRET signal generated from hybridomas encapsulated in picodroplets and screened for secretion of human TNF-alpha specific IgG. Image Credit: Sphere Fluidics
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About Sphere Fluidics
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.
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.
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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