Introduction
Antibody-based biologics have become an important class of modern medicine since the launch of the first therapeutic monoclonal antibody (mAbs) in 1986. More than 80 therapeutic mAbs have been approved for clinical use, and the rate of FDA-approved mAbs continues to increase. Yet, traditional semi-automated technologies used to develop mAbs are time-consuming and resource-intensive. This results in inefficient discovery and development timelines because of their low screening throughput and impedes the ability to deep mine B cell repertoires effectively.
To accelerate antibody discovery and development, leading biopharmaceutical companies are applying powerful B cell screening technologies, like Sphere Fluidics' Cyto-Mine®, that allow the highly efficient isolation of cells that produce potential therapeutic antibodies. Leveraging picodroplet-based technology, Cyto-Mine® offers a robust, high throughput platform for screening and isolating rare antigen-specific B cells directly from large heterogeneous populations in just two days. By using Cyto-Mine®, researchers can overcome challenges in the antibody discovery process and significantly shorten the antibody discovery workflow.
What are the current challenges in antibody discovery workflows?
In antibody discovery, the generation of early candidates typically starts with a known, validated target where candidate antibodies need to be generated for ‘Hit’ selection. Traditionally, animals, whether they are regular or transgenic mice, rats or rabbits, are immunized to stimulate an immune response, and after several weeks B cells can be harvested and fused with myeloma cells to create hybridoma populations. Alternatively, the B cells can be isolated directly but, whichever process is adopted, the cells producing the antibodies require extensive screening and characterization. This is where bottlenecks typically occur when using conventional screening methods such as flow cytometry and ELISA.
Flow cytometry is a high throughput method for screening large cell populations, however, it comes with some significant limitations. The process of flow cytometry is harsh and can cause damage to any cell including those that are rare or valuable, therefore reducing cell viability and potential recovery. Additionally, flow cytometry cannot be used to readily measure cell secretion, flow cytometers offer cold capture secretion assays where the secreted molecules are retained at the cell surface using cell manipulation but any secreted molecules not captured diffuse into the extracellular environment.
Once the antibodies have been screened and characterized the cells then need to be subcloned into monoclonal populations. This can be done using semi-automated technologies such as cell printers and cell-in-well imagers, adding multiple instruments into the workflow, in turn making the whole process very time consuming and labor-intensive.
What are picodroplets and how do they overcome these challenges?
Sphere Fluidics’ Cyto-Mine® Single Cell Analysis System, harnesses proprietary picodroplet-based technology to encapsulate and rapidly screen antibodies directly from whole B cell repertoires or hybridoma populations while preserving cell viabilities. Using this robust and high-throughput method, researchers can identify antibody-secreting cells and isolate rare cells secreting antigen-specific antibodies in just 1-2 days.
Picodroplet microfluidics is based on the encapsulation of a single cell within a picolitre volume water-in-oil emulsion droplet 'picodroplet.' Picodroplets supply a unique microenvironment for every cell, where secreted biomolecules and proteins can be trapped and assayed. Encapsulation in picodroplets ensures that concentration of the molecules secreted by the cell increases extremely quickly, and protects single cells from shear stress, thereby overcoming some of the major barriers of flow cytometry and fluorescence-activated cell sorting.
Highly Concentrated
As briefly mentioned, one of the advantages of conducting assays in picodroplets is that picodroplets are around 5-6 orders of magnitude smaller than volumes in conventional assays. So the concentration of secreted antibodies from a single cell in a picodroplet is approximately 5-6 orders of magnitude bigger, leading to a highly concentrated assay that utilizes a fraction of the amount of reagents overall optimizing efficiency and shortening assay time dramatically.
One in a Million
In addition to single cell encapsulation, numerous cells may be captured within a picodroplet, giving the benefit of being able to screen tens of millions of cells in a single run. Screening picodroplets with numerous cells an initial assay (to identify cells secreting antibodies) can be performed before a secondary screen where the 'hit' cells are re-encapsulated and assayed for antigen specificity. This method of two-step screening with Cyto-Mine® provides the capability to locate an antigen-specific population of B cells, which can have "hit cells" happening at rates as low as 0.0005%.
As few B cells survive the fusion process with myeloma cells, microfluidic-based picodroplet technology supplies a considerable advantage over the resource-intensive process of hybridoma generation. Employing a fully automated and integrated platform allows the direct screening of B cells, not affecting their viability, not needing cell fusion, and heightening the probability of identifying that valuable cell.
Cell-Friendly
The cell-friendly environment of the picodroplet is one other key advantage. When a picodroplet forms, it encapsulates a single cell with culture medium which supplies a protective 'capsule,' which supports cell integrity as they travel through the microfluidic channels. The picodroplet shields cells against shear stress (like a cushion) and the bio-compatible surfactants utilized in picodroplet formation promote high levels of oxygen concentration and gas exchange.
Picodroplet generation
How can you get started using picodroplet-based technology?
Combining B cell isolation, screening, and antigen-specific assays into a fully automated workflow, Cyto-Mine® enhances the screening efficiency and decreases the costs of assay reagents. By addressing the critical requirements of lower costs, higher throughput, and greater sensitivity, this powerful technology is poised to revolutionize antibody discovery, providing solutions to transform antibody discovery and the development of novel biotherapeutics. For more information, visit: spherefluidics.com
References
- Ecker, D., Jones, S. D. & Levine, H., 2015. The therapeutic monoclonal antibody market. BioProcess Technology Consultants, Inc., pp. 9-14.
- Lu, Z.-J.et al., 2012. Frontier of therapeutic antibody discovery. World Journal of Biological Chemistry, pp. 187-196.
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 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.