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Monoclonality

Document monoclonality – a regulatory metric for therapeutic cell lines

What is monoclonality?

Monoclonality is term that describes a cell line that originates from a single progenitor (single cell) - and is therefore monoclonal. Cell line development and assurance of monoclonality are critical steps in the process of generating biopharmaceutical molecules, such as monoclonal antibodies. A cell line can be established following the isolation of a single viable cell robustly expressing the protein of interest. A key milestone in this process is documenting evidence of clonality to ensure the genetic reproducibility of the cell line.

Documentation of monoclonality is a regulatory metric for therapeutic cell lines and is typically image-based, whereby an image of a single cell is recorded and included in regulatory filings.

Monoclonality Verification - viewing origin of a colony

Verification of monoclonality—viewing the origin of a colony. The growth (image) history of each well can be tracked back to its starting point—providing evidence of monoclonality

Why is monoclonality important?

As regulations for cell line development become increasingly more stringent, researchers will be required to perform single-cell cloning and provide evidence that a cell line is derived from a single cell—proof of clonality. Traditional cloning methods (e.g. limiting dilution and FACS) use statistical analysis to determine a confidence level for monoclonality. However, the documentation of monoclonality has driven the need for more robust technologies and methodologies in bioprocessing. Many researchers now routinely use imaging systems, such as the CloneSelect Imager, to verify monoclonality and monitor cell growth in cell culture media.

High assurance monoclonality for cell line development

The development of cell lines that express a specific protein of interest is critical to the generation of biologics, and regulatory agencies require evidence of monoclonality in order to get a biologic to the marketplace. The traditional approach involves visualization of wells using transmitted white light on day 0 to confirm the presence of a single cell. However, this approach is not without challenge, as cellular debris and well artifacts can be easily mistaken for cells. Consequently, cell line developers typically evaluate cells at the colony state and trace back the origins of the colony to confirm monoclonality.

The workflow highlights the process of CloneSelect Imager System to verify monoclonality based on objective image analysis. Coupled with the CloneSelect Single-Cell Printer for single cell sorting improves the workflow with both an increase in clonal outgrowth and additional image-based assurance of monoclonality.

Monoclonality Workflow
  1. Sort single cells into microplate wells – Single, viable cells must be isolated and cloned in order to ensure that the cell population is genetically identical, significantly reducing the heterogeneity of expression.

  2. Scan plate WL at Day 0 and subsequent days – Image plate at Day 0 and subsequent days. Documentation of monoclonality (a regulatory metric for therapeutic cell lines) is typically image-based, whereby an image of a single cell is recorded and included in regulatory filings.

  3. Review plate scan and assess overall outgrowth (WL) – Review and analyze images tracking cell growth rates, confluence measurements, and colony formation.

  4. Confirm monoclonality and export well data for regulatory submission – Confirm monoclonality and export well data in detailed report for regulatory submission.

Improve clonal outgrowth with single-cell sorter

The CloneSelect Single-Cell Printer Series is a fully automated system that utilizes proprietar microfluidics-based technology and real-time image analysis to sort and deposit single cells into standard microplates—while simultaneously providing assurance of monoclonality through image documentation. It addresses the shortcomings of limiting dilution and flow cytometry-based cell sorting by offering image-based evidence for clonality, fast and efficient single-cell deposition based on white light (c. sight and f. sight systems) or optional fluorescence (f. sight systems only), excellent post-deposit cell growth, minimal risk of cross-contamination, and extremely low cost for maintenance. When coupled with the CloneSelect Imager, the Single-Cell Printer series demonstrates improvements over both of these workflows with both an increase in clonal outgrowth and additional image-based assurance of monoclonality.

Improve clonal outgrowth with single-cell sorter

The CloneSelect Single Cell Printer Technology(A) After a cell-containing sample is loaded into cartridge, the cartridge is mounted onto the SCP and a print run can be initiated. During
the printing procedure droplets are continuously generated by a piezo-driven actuator in a predictable sequence. Droplets that contain no cells or more than one cell are siphoned away using a vacuum
mechnism directly below the nozzle. When single-cell events are identified, a shutter mechanism inhibits the vacuum allowing the single cell-containing droplet to fall into the well. (B) When a single
cell event is detected, five images are captured to provide evidence of clonality at the nozzle. Images 1-3 show the presence of a cell prior to droplet ejection, image 4 marks the region containing the
single cell, and image 5 shows nozzle after droplet ejection.

Microplate imaging, clone characterization and report generation Microplate imaging, clone characterization and report generation

(A) Day-10 clones derived from SCP printing session are imaged. The Plate Thumbnails tab allows visualization of colonies. Clicking on individual wells navigates to the user to the View Image tab, where a whole-well image is shown. (B) The Loci Count tab allows counting of colonies to be optimized by adjusting the area and compactness, a measure of roundness, of a colony. (C) In the Generate Report tab, cell regions (green boxes) can be designated and adjusted for each time point in a series to characterize the growth from a single cell to a colony.

What is monoclonal and what is not

By using the CloneSelect Imager software to zoom in on individual, immobilized colonies, it is possible to track growth and determine whether a colony is monoclonal by reviewing the colony images at different time points. In figure below, the images of two colonies are shown. By observing growth of the two colonies and tracking the images back to Day 0, it can be determined whether or not the colony originated from one or more cells. The addition of the beads (shown in the yellow circles) to the semi-solid media serves as a location reference to confirm that the same colony is being imaged each time.

Monoclonal One-cell Colony

CHO-s cell growth in CloneMedia CHO Growth A semi-solid mediaCloneSelect imager was used to capture images from the 6-well plates at multiple time points. On day 0, it is clearly observed on the top row that one cell is present while on the bottom row, two cells are observed. The yellow circle show the position of a bead that serves as a location reference to confirm that the same colony is imaged over time.

Providing image evidence of monoclonality (report)

Providing image evidence of monoclonality in the cell line development process is not as simple as exporting an image of a single cell. For example, high-resolution images of the entire well should also be inspected to ensure the absence of a second cell.

With a few simple clicks, the Monoclonality Report feature on the CloneSelect™ Imager (CSI) objectively organizes the supporting image evidence needed to establish clonality into an easily shareable report, saving researchers hours typically required to do the same process manually.

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Verification of monoclonality

Methods for monoclonality assurance

Learn more about the different methods to verify and document monoclonality.

  • Confident assurance of clonality using calcein AM

    Confident assurance of clonality using calcein AM

    Confident assurance of clonality using calcein AM with minimal effect on viability

    Here, we demonstrate an optimized workflow using the fluorescence reagent, calcein AM, in conjunction with a fluorescence-capable CloneSelect™ Imager that shows similar viability to label-free conditions while simultaneously providing high assurance of clonality.

    Read application note  

    eBook: Optimize your high value cell lines

    Optimize your high value cell lines

    In this eBook, we present an overview of the cell line development workflow as well as high throughput solutions for accelerating the process, and enabling easier and faster selection of high-producing mammalian cell lines.

    Download eBook  

  • Fluorescent method for identifying monoclonal CHO-S cells

    Fluorescent method for identifying monoclonal CHO-S cells

    Rapid monoclonality verification methods to boost cell line development

    While monoclonality verification via white light imaging is possible, debris, dust, and air bubbles make it difficult and time consuming to identify single cells which may cause high value clones to be discarded. Here we present a fluorescent method for identifying monoclonal CHO-S cells using celltracker green cmdfa.

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    High assurance monoclonality for cell line development

    High assurance monoclonality for cell line development

    Limiting dilution or fluorescence activated cell sorting is typically performed to seed single cells into a well. Microscopy is then used to determine the number of cells seeded in each well and monitor cell growth. While monoclonality verification via white light imaging is possible, debris, dust, and air bubbles make it difficult and time consuming to identify single cells which may cause high value clones to be discarded

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  • Identify monoclonal CHO-S cells Grown in semi-solid media

    Identify monoclonal CHO-S cells Grown in semi-solid media

    Confident identification of monoclonal cho-s cells grown in semi-solid media

    Growing CHOcells in CloneMedia CHO Growth A is a more ecient way to clone CHO cells than performing limiting dilution in liquid media. Semi-solid media immobilizes the cells which prevents the cells from moving during routine handling. This enables researchers to confidently track the growth of a single cell into a colony.

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    Improve cloning efficiency with higher assurance of clonality

    Improve cloning efficiency with higher assurance of clonality

    A workflow combining single-cell isolation and microplate imaging improves cloning eciency with higher assurance of clonality

    Optimized workflow saves time, labor and reduces consumable and reagent costs by increasing outgrowth efficiency, while providing higher assurance of clonality and rapid documentation that is ready to use for submission to regulatory agencies.

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  • Monoclonality reporting

    loneselect Imager Monoclonality report feature

    Cloneselect imager monoclonality report feature

    With a few simple clicks, the Monoclonality Report feature on the CloneSelect Imager objectively organizes the supporting image evidence needed to establish clonality into an easily shareable report, saving researchers hours typically required to do the same process manually.

    View brochure  

    Simplify screening and selection of mammalian cell lines

    Simplify screening and selection of mammalian cell lines

    Using ClonePix System to assess monoclonality

    The ClonePix System system is a powerful tool for rapid screening and isolation of secretory mammalian cell lines typically used in the production of therapeutic proteins and research monoclonal antibodies. The data presented in this tutorial supports the idea that picking colonies of mammalian cells from semisolid medium is an effective means to isolate clonal cell lines.

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Resources for Monoclonality

Videos & Webinars

Tips to automating molecular cloning and strain engineering applications

Monoclonality Workflow

Monoclonality Workflow