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Antibody development for biotherapeutics Select optimally secreting cell linesExample: select optimally secreting mAb clones
Imaging principle
Case study: Development of cell lines for biotherapeutics“ClonePix FL has great advantages in selecting optimal producer cell lines and shortening timelines for early stage cell line development” Dr. Jianguo Yang, Group Leader in Cell Line Development, MedImmune
Development of ClonePix FL methods for NS/0 and CHO |
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ClonePix FL
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Limiting dilution
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Comparison of cloning methods
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Limiting dilution
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ClonePix FL
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Comparison of top clones from ClonePix FL and limited dilution
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High titers of final clones from ClonePix FL method
CHO cells - 14 day fed batch process in shake flasks |
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Conclusion from MedImmune: ‘ClonePix FL is a powerful tool in cell line development. This method makes selecting the optimal producers faster and less labour intensive and shortens cell line development time.’
Further information
- Selection of high producing mammalian cells secreting therapeutic proteins / peptides
Eliminate unstable clones
Since lack of stability can be a problem with early stage transfectants, ClonePix systems can be used reveal clonal instability.
ClonePix can re-plate aliquots of selected clones into semi-solid media and, within 4-7 days, re-image to verify and compare production rates of the daughter clones and/or re-screen for sub-clones within 7-14 days. Potential instability is indicated by varying levels of fluorescence between the daughter clones.
Example shows results from a selection of stable clones by second round screening
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Top 2% of transfected population of suspension- adapted CHO cells collected and assayed for productivity
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Productivity vs. fluorescence after second round screening. Note elimination of high fluorescence, low productivity (unstable) clones
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Cell confluence and cell number can be tracked by CloneSelect Imager.
Antibody discovery for research
Screen and select antigen-specific hybridomas or B cells
“We have implemented the use of ClonePix FL to increase our fusion productivity by approximately 50%, while decreasing our time from fusion to stable clone by 50%.”
Dr Robin Barbour, Associate Director Elan Pharmaceuticals
Example: screening antigen-specific hybridomas
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Screen and select antigen-specific clones in situ
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Suitable for a broad range of antigens
- - 160kD multimeric protein to 2.6kD phosphopeptide
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White light image
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FITC conjugated 60kD antigen-specific IgG producing clone
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White light image
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FITC conjugated 60kD control protein
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Imaging principle
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Label-free detection of secreted antigen-specific MAbs
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Further information
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Hear more in a customer's words:
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Isolation of antigen-specific clones from hybridoma fusions
Protein expression and production
Select by intrinsic reporter expression
Example: detection of intrinsic GFP reporter
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Human breast cancer cells (MCF-7) transfected with internally expressed GFP-fusion protein
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Cells grown as adherent colonies in MEM Earle’s liquid medium + 10% FBS
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Day 8: Clones expressing highest GFP selectively picked
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White light
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GFP
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After picking
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Select by cell surface expression marker
Example: detection of cells expressing P2Y1 receptor
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Further information
- Selection of mammalian cell colonies by cell surface protein expression
Select secretors of a tagged recombinant
Example: detection of secreted monomeric His/FLAG-tagged protein
- Adherent CHO cells stably transfected
- Grown as suspended colonies in CloneMatrix-based semi-solid medium with FITC-conjugated anti-His and anti-FLAG polyclonals
- High secretors screened and picked at day 8
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White light image
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FITC image
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Imaging principle
Stem cell screening/Maintenance of stem cell lines
Screen by detection of surface marker expression
Example: automate maintenance of undifferentiated mouse embryonic stem cells (mES)
- Efficient propagation of clonal stem cells using markers for pluri-potency
- Screen against differentiated cells
- Facilitate genetic modification assays – detect expression of inserted or deleted genes
For mouse embryonic stem cell (mESC) culture, propagation and cell line development, the high resolution and selectivity of enables the most pluri-potent colonies to be picked from clonal monolayers and clearly discriminates between differentiated and undifferentiated clones.
Example shows imaging of CGR8 mES cells after 4 days growth using rhodamine-conjugated anti-SSEA1. Using antibody to discriminate colonies of pluri-potent stem cells based on cell surface receptor expression has no effect on the lineage of differentiation of these cells post-picking, giving good outgrowth of undifferentiated cells.
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Pluripotent cell colonies show high expression of SSEA1 and small, round morphology
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CGR8 growth in CloneMatrix/GMEM semi-solid media
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CGR8 colony post pick
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Further information
- Selecting clones of mouse embryonic stem cells
Maintain lines utilizing colony morphology (white light)
Example: maintenance of human stem cells (BG01V hES) utilizing white llight and morphology
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Stem cell clones shown by white light imaging on ClonePix
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Typical picked clone collected to 96-well plate. Imaged on CloneSelect Imager
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Brochures
ClonePix™ 2
Screening and selection of mammalian clones
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Accelerate Cell Line Development
ClonePix with CloneSelect Imager
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Recorded Webinars
Using high throughput automated selection to increase discovery of rare, high producing mammalian cell lines
Dr Dharshanan presents how the use of ClonePix has increased the probability of finding rare high producing clones at Inno Biologics.
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Listen now >
Optimal selection of monoclonal cell lines producing high amounts of biopharmaceutical human IgGs
Dr Gerritsen, Technology Expert, explains how Genmab have optimized and integrated emerging technologies to achieve an optimal, state-of-the-art process for generation of CHO cell lines expressing therapeutic IgGs.
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