This article explores the evolving role of high-throughput picodroplet technologies in drug discovery by highlighting their use in bispecific antibody development, cell line engineering and cell therapy research.
Microfluidic systems based on picodroplet technology provide a high-throughput and sensitive method to perform quantitative screening assays by encapsulating single cells in picolitre volume compartments.
This single-cell approach enables researchers to gather valuable information on the functional capacities of individual cells that cannot be obtained from large population-based assays.
These microfluidic systems can encapsulate single cells at the rate of thousands of droplets per second in water-in-oil picodroplets that act as miniature reaction chambers.
Cell-secreted molecules are trapped in picodroplets and accumulate quickly to a concentration that can be reliably and quantitatively detected using assays based on the Förster resonance energy transfer (FRET) theory.
In a FRET assay, two fluorescent probes, donor and acceptor, will bind to the target secreted molecule. The binding event will bring them into proximity, resulting in energy transfer from donor to acceptor.
This energy transfer can be detected as a decrease in the donor’s fluorescent signal and a corresponding increase in the acceptor’s one. The droplets containing desirable cells are sorted by fluorescence and dispensed into individual wells in a microtitre plate for downstream analysis.
Here, we present some of the most exciting potential applications of picodroplet technology for biopharmaceutical discovery.
Bispecific antibody production
Effectively enrich single-cell clones with high productivities: Bispecific antibodies (BsAbs) are molecules designed to bind to two distinct epitopes or antigens.
Owing to their improved therapeutic efficacy compared with conventional monoclonal antibodies (mAbs), they are gaining a lot of interest in the biopharmaceutical industry, especially in cancer immunotherapy and the treatment of inflammatory diseases.
The development of BsAbs requires stable and high-yield cell lines that produce improved concentrations of correctly assembled bispecifics. However, the selection of high-producing cell lines is usually time-consuming and labour-intensive.
Conventional assays used to detect and characterise BsAbs produced by cells, such as ELISA, LC-MS and SE-UPLC, are low-throughput and relatively expensive.
Also, the product profiles of BsAbs are much more complex, consisting of a mixture of single peptide chains, monospecific half molecules, monospecific homodimers and bispecific heterodimers, necessitating the screening of many clones to ensure that the cell lines producing the best quality molecules are obtained.
Picodroplet-based technologies combine high-performance assays with efficient single-cell cloning to transform traditional BsAbs development workflows.
One of the unique benefits of working with FRET is that it enables flexible assay design and can be customised to detect both distinct bispecific antibody properties simultaneously, thus allowing the rapid detection and selection of single cells that produce intact bispecific antibodies.
When combined with picodroplet technology, researchers can capture single cells and all the factors secreted by those cells in a picodroplet. The miniaturised volumes enable secretions to reach a detectable concentration quickly; the secretion profile can then be analysed to evaluate the quality of the products from each cell clone.
Assay miniaturisation in picodroplets also increases screening throughput, enabling a large cell population to be rapidly screened and potential development clones to be established in just one day.
This may significantly reduce the number of clones that need to be tested downstream to decrease overall project timelines and cell line development resources for BsAbs.
Genome editing
Isolate genome-edited single cells with picodroplet encapsulation: Genetic engineering is an established R&D tool with promising applications, especially to treat cancer and genetic diseases. CRISPR/Cas9 technology, a powerful form of genetic engineering, has now transformed the range, precision and efficiency with which genomes can be modified. However, the production and testing of gene-edited cell lines remain inefficient.
Typical genome editing experiments begin with the delivery of Cas9 in the form of DNA, RNA or a protein complex — together with gRNA — and, in the case of gene insertion or mutation, the addition of template DNA.
Current gene delivery approaches, such as electroporation, viral transduction and lipid-mediated transfection, are slow, inefficient and often imprecise, requiring high manual handling, data collection and decision making.
The next step in the CRISPR experiment involves transitioning from a heterogeneous cell pool to a single cell clone. This consists of the isolation and expansion of individual clones with the desired modifications and the screening of the desired phenotypic changes for each clone.
Conventionally, researchers conduct single-cell isolation by limiting dilution or flow sorting and then use different methods for the downstream analysis and validation of clones, such as next-generation sequencing and microscopy.
Overall, this process, using multiple tools and manual steps that require additional wet lab work, is slow and laborious.
Picodroplet technology bypasses inefficient gene delivery and manual handling inconsistencies with “hands off” automation by allowing researchers to encapsulate cells with the gene delivery and editing reagents in a picodroplet to encourage single-cell transfection.
Picodroplet compartmentalisation enables cells to be designed on a single cell basis in a controlled environment in which the local concentration is much smaller. This minimises diffusion and eliminates the bias and inefficiencies seen with transfection treatments on a bulk cell population.
The combination of microfluidics and technology also enables large numbers of cells to be processed in a single experiment and facilitates easier detection of low-frequency events; as such, transfected cells can be rapidly selected and dispensed into microtitre well plates.
In addition, once encapsulated in picodroplets, cells maintain good viability, ensuring single-cell quality for growth. These capabilities all contribute to a significant reduction in the amount of work done for downstream analysis and validation.
Cell therapy research
Validate the functionality of rare, antigen-specific T-cells: During the last few years, there has been increasing interest in cell immunotherapy as a strategy to harness the immune system to fight tumours, such as the reprogramming of T-cells collected from patients to produce personalised medicines.
T-cell therapy capitalises on the body's immune system to kill tumour cells by extracting and manipulating the patient's T-cells to attack cancer cells.
Assays capable of characterising engineered T-cells to identify the drivers for the desired phenotypes, such as polyfunctionality, serial killing and proliferation, are essential for developing novel cell therapies.
However, our ability to measure the characteristics of T cells of interest and to select the most effective cells for therapy is limited by the low efficiency of ELISPOT and flow cytometry based methods.
The advantage of picodroplet technology is that it enables the isolation of a single cell in a picolitre droplet to perform assays to detect and quantify molecules, such as cytokines, secreted by a single T-cell that can be used to validate the T-cell engineering process.
It is also possible to encapsulate a T-cell with a tumour cell to assess and confirm the potency of the engineered T cells for tumour cell killing.
In summary, these are just some of the applications of picodroplet-based microfluidic technology for biopharmaceutical discovery; there is more to come. Picodroplet technology is revolutionising single-cell analysis with unprecedented throughput, precision and control.
It is already promising in several areas, particularly drug discovery, with applications in bispecific antibody development, cell line engineering and cell therapy research.
Reference
- D. Josephides, et al., “Cyto-Mine: An Integrated, Picodroplet System for High-Throughput Single-Cell Analysis, Sorting, Dispensing and Monoclonality Assurance. SLAS TECHNOLOGY: Translating Life Sciences Innovation (2020): https://doi.org/10.1177/2472630319892571.