Accelerate Your Antibody Discovery and Cell Line Development Workflows with Cyto-Mine

Transcription

1 Accelerate Your Antibody Discovery and Cell Line Development Workflows with Cyto-Mine

2 Cyto-Mine The Single Cell Analysis Platform Cyto-Mine is the world s first fully integrated platform for high-throughput single cell analysis based on microfluidic picodroplet technology. By encapsulating single cells in picolitre volume droplets, Cyto-Mine enables the rapid analysis, sorting, imaging and dispensing of single cells into individual wells of microtitre plates, reducing both time and costs compared to traditional approaches.

3 Cyto-Mine Key Features Integrated microfluidics Single cell secretion assays Uses microfluidic picodroplets Fully integrated workflow Allows single cell secretion assays Validated for CHO cells, hybridomas and B cells High throughput screening Fluorescence-based sorting 10,000 s~40,000,000 cells/run/day Monoclonality assurance High fidelity dispensing into 96- or 384- well plates Image-based verification of monoclonality Cell friendly workflow Consistently high cell viability High single cell out-growth rate Designed for biopharma Used in antibody discovery and cell line development Sterile, single use consumables

4 Cyto-Mine Overview Encapsulate Assay Sort Image & Dispense cells Encapsulate up to 200,000 single cells or 40 million cells in pools Screen cell populations by antigen specificity or immunoglobulin productivity Sort based on fluorescence intensity to find positive hit cells or highest producers Image individual picodroplets during dispensing into 96- or 384-well plates to record monoclonality of single cell occupancy

5 Fully Integrated Workflow Cyto-Cartridge Sample Loading Encapsulation Incubation Assay Sorting Dispensing The Cyto-Cartridge delivers: Sample Loading easy-to-use, single touch point Encapsulation cells are captured in picodroplets stabilized by biosurfactants Incubation & Assay incubation at 37 o C allows for protein secretion followed by a fluorescence-based assay Sorting picodroplets are sorted based on fluorescence intensity Imaging picodroplets are imaged multiple times during dispensing (can verify monoclonality) Dispensing encapsulated cells can be dispensed into 96- or 384-well plates

6 Cyto-Mine Workflow Sample Loading Sample Loading Sample Loading o Sterile, single use Cyto-Cartridge o Animal origin free materials and reagents o 1mL (Cell suspension + Assay reagents) o 10,000 s ~ 40,000,000 cells per run o Compatible with most culture media Cells and assay reagents are pipetted into the Cyto-Cartridge. o Validated sample type: CHO cells, B-Cell, Hybridomas, T cells, HCT

7 Viability (%) Cyto-Mine Workflow Encapsulation Encapsulation Cells + Assay Biosurfactant Cell viability (CHO) over time in picodroplet pl pl droplet Hours Cell Encapsulation in Picodroplets Cyto-Mine Generates millions of picodroplets within an hour ~ 300 pl picodroplets Generated using biosurfactants Supports a high percentage of monoclonality Supports both single and multiple cell loading modes for even higher throughput

8 probability (%) Cyto-Mine Workflow Encapsulation Single cell mode Encapsulation Theoretical Cell Number Distribution In Picodroplets E cells/ml cells/ml Number of cells in a picodroplet

9 probability (%) Cyto-Mine Workflow Encapsulation Single cell mode Encapsulation Theoretical Cell Number Distribution In Picodroplets Multiple cell mode cells/ml cells/ml E cells/ml 1E cells/ml Number of cells in a picodroplet

10 Cyto-Mine Workflow Assay Incubation Incubation chamber in Cyto-Cartridge Rapid antibody detection. The size of the picodroplets generated in Cyto-Mine is only several hundreds of picolitres in volume, about 5-6 orders of magnitude lower than the volumes in conventional assays. This means, given the sample incubation time, the concentration of secreted antibodies from a single cell in picodroplets is 5-6 orders of magnitude higher than in conventional assay. Quantitative measurement of antibody secretion from single cells. In Cyto-Mine, it only takes hours incubation time before the system can detect antibody secretion from each of the encapsulated cells. Quantitative measurement of antibody secretion in comparison to bulk culture. Cells secrete at comparable rates between the two methods.

11 Cyto-Mine Workflow Assay FRET Assay in Picodroplets Assay o FRET = Förster Resonance Energy Transfer o Dynamic Range: 0.1 s ~ 100 s nm o Short incubation time before detection ( min) o Allows quantitative detection and differentiation of low and higher producers o Antigen-specific assays are easily developed Encapsulating single cells in picodroplets enables detection of molecules secreted by the cells (rather than cell surface-bound molecules). Sphere Fluidics has developed a panel of homogeneous FRET assays which allows detection of IgG from various species inside picodroplets. The homogeneous assays can provide information on the endpoint concentration of the antibodies in the picodroplets, essentially, enabling single cell sorting and cloning based on ranking their productivities. o Validated IgG species: Human, Mouse, Rat, Rabbit o Animal component free assay reagents available o Assays can be custom developed

12 Cyto-Mine Workflow Assay Assay Response Curve (plate reader) FRET Assay in Picodroplets o FRET = Förster Resonance Energy Transfer o Dynamic Range: 0.1 s ~ 100 s nm o Short incubation time before detection ( min) o Allows quantitative detection and differentiation of low and higher producers o Antigen-specific assays are easily developed o Validated IgG species: Human, Mouse, Rat, Rabbit o Animal component free assay reagents available o Assays can be custom developed

13 Acceptor Fluorescence Acceptor Fluorescence Acceptor Fluorescence Cyto-Mine Workflow Assay Standard Plot CHO Secretion High Conc. 5 mg/l 2 mg/l 1 mg/l 5 mg/l 2 mg/l 1 mg/l Fast secretors 0.5 mg/l 0.5 mg/l Secretors 0.1 mg/l 0.1 mg/l 0 mg/l Low Conc. 0 mg/l Empties Non-secretors Donor Fluorescence Donor Fluorescence Donor Fluorescence The above scatter plot shows results of detection of a library of picodroplets containing different concentrations of target IgG. Picodroplets containing assay reagents and 0, 0.1, 0.5, 1, 2, 5 mg/ml target IgG were generated separately, then mixed at an equal ratio and analyzed by measuring Laser induced fluorescence. In a Cyto-Mine instrument run, the user can gate and sort a (sub-)population of picodroplets by manually drawing a region of interest on the scatter plot. From 10,000 up to 40,000,000 picodroplets can be analyzed in a single Cyto-Mine experiment run.

14 Cyto-Mine Workflow Sorting Sorting Cell Sorting in Picodroplets o Sorting based on fluorescence signal o One laser, two color detection o >1 million picodroplets/h o Minimum or no damage to cell viability o Superior single cell out-growth rate Flow sorter

15 Cyto-Mine Workflow Dispensing Dispensing Single Picodroplets Dispensing o Gently dispense 1Hz o High precision and fidelity (>99%) o Fluorescence measured again at dispensing o Images of picodroplet taken before dispensing o Five snapshot images of cell(s) in each picodroplet o Monoclonality analysis by software

16 Cyto-Mine Workflow Dispensing Flow Direction Dispensing A B C D E F G H Cyto-Mine validates single cells (green) and alerts attention to picodroplets that are empty (yellow) or containing two or more cells(red).

17 Challenges in Discovery and Development Discovery of cells that produce antibodies against the target Development of cell lines to make grams-kilograms of antibodies Challenges in Antibody Discovery o o o o High-throughput technology is needed to screen whole B-cell repertoires or hybridoma populations. Deep B-cell repertoire interrogation requires high throughput single cell technologies. Flexibility in assay design is needed. High sensitivity and specificity is required to find rare antibodies with desirable characteristics. Challenges in Cell Line Development o o o o o Need for screening of large panels of single cells to select only the high producers. Lack of assays reliably ranking and selecting based on single cell productivity. Cell friendly processing is desirable to allow improved cell out-growth. Evidence of monoclonality is a regulatory requirement. Pressure to minimise development time lines. High throughput screening Single cell secretion assay Monoclonality Assurance Cell Friendly Workflow Designed for biopharma Integrated Microfluidics

18 Cyto-Mine Greatly reduces timelines Increases screening capabilities Delivers confidence in monoclonality Cyto-Mine Cyto-Mine Software Suite Cyto-Cartridge Consumable Cyto-Surf Consumable

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