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. 대상 단백질을 로버스트하게 발현하는 생존 가능한 단일 세포를 분리한 뒤, 세포주를 확립할 수 있습니다. 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.
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.
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.
Scan plate WL at Day 0 and subsequent days – Image plate at Day 0 and subsequent days. Monoclonality를 문서화(치료 세포주의 규제 지표)하는 것은 일반적으로 단일 세포의 이미지를 기록하고 이를 규제 문서 파일에 포함하는 이미지 기반 작업입니다.
Review plate scan and assess overall outgrowth (WL) – Review and analyze images tracking cell growth rates, confluence measurements, and colony formation.
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.
Microplate imaging, clone characterization and report generation
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.
Providing image evidence of monoclonality (report)
Cell line development 과정에서 monoclonality 형성능의 근거를 이미지로 제시하는 것은 단일 세포의 이미지를 추출하는 것처럼 간단하지 않습니다. 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.
Methods for monoclonality assurance
Learn more about the different methods to verify and document monoclonality.
Resources for Monoclonality
Double confirmation of clonality. Image based evidence confirming single cell deposition event, and single cell in 96- and 384-well plate post sort
A workflow combining single-cell isolation and microplate imaging improves cloning efficiency with higher assurance of clonality
단일 세포의 분리는 단일 세포 유전체학부터 항체 발굴 및 Cell line development에 이르는 광범위한 응용 분야와 관련됩니다.
특정한 대상 단백질이 발현된 세포주의 개발은 단일클론항체와 같은 생체의약품 분자 생성에 중요합니다. ...세포를 확립하기 위해서는...
여기서는 CellTracker Green CMDFA를 이용하여 단일클론 CHO-S 세포를 식별하기 위한 형광 방법을 제시합니다.
The ClonePix System was developed to overcome many of the drawbacks typically associated with isolating choice mammalian cell clones, during screening and selection of secretory cell lines…
Confident Identification of Monoclonal CHO-S Cells Grown in Semi-Solid Media using the CloneSelect Imager
생물학적 약물를 승인받기 위한 규제 기준은 지속적으로 높아지고 있습니다. 생물학적 세포주에 대한 규제 지표 중 하나인 monoclonality의 디지털 문서화는...
동영상 및 웨비나