Automatically screen more clones in less time than conventional techniques, select cells with optimal expression levels, and pick colonies with accuracy with the ClonePix™ 2 System. ClonePix Systems are now used in over 100 laboratories around the world to increase workflow productivity, leaving more time to better characterize target proteins and run new projects. A large number of biopharma companies have implemented ClonePix Systems in their routine use and data are cited increasingly in scientific publications and conferences

  • Select cells with optimal expression levels with improved ranking consistency. Increase probability of finding optimal producers and select cells 
  • Cut cell line/antibody development times – avoid limiting dilution
  • Reject poor performers at an early stage based on expression levels in situ
  • Pick colonies with accuracy and confidence. Reduce risk of colony disturbance with robotics redesign.
  • Improve workflow efficiency with fewer plate handling steps

Antibody Development for Biotherapeutics

Select Optimally Secreting Cell Lines

Select Clones Secreting Optimal Monoclonal Antibodies

  • Screened 5463 colonies (5 plates) using white light and fluorescent imaging
  • Selected 16 high secretors
  • CHO-S cells grown in CloneMedia-CHO
  • Screened for IgG secretion by addition of CloneDetect agent
  • Clones ranked according to fluorescence
    • User defined selection parameters
  • System accurately picks clones for expansion
    • Proximity settings avoid picking neigboring clones
    • System transfers each colony into one well of a 96-well microplate 
CHO-S expressing human IgG: stiched image
of PetriWell-1 plate picked on day 13
Ranking plot - Clones ordered by
fluorescence intensity (secretion)

Imaging Principle - Label-Free Detection of Secreted Antibody

image_mAb.gif

  • Secreting clones grown in semi-solid media
  • CloneDetect agent added
  • Secreted antibodies form fluorescent precipitate

Development Of Cell Lines For Biotherapeutics

Select Optimal Producer NS/0 and CHO Cell Lines Using ClonePix System

  • Screens more clones than traditional methods, increasing the probability of finding rare, high secretors
  • Automatically isolates clonal colonies, removing the need for limiting dilutions
  • In situ indication of high titer cell lines removes unwanted clones from further processes

COURTESY OF DR. JIANGUO YANG, GROUP LEADER IN CELL LINE DEVELOPMENT, MEDIMMUNE LLC

Comparison of Techniques:
 

case_study_1.gif

ClonePix System

  • From parental cell line to shake flask: 1 month
  • 10000 clones screened

Limiting dilution

  • From parental cell line to shake flask: 2 months
  • 1000 clones screened
Comparison of Cloning Methods:
 

case_study_2.gif

ClonePix System

  • 2500 clones per week
  • 1 week cloning

Limiting dilution

  • 300 clones per week
  • 3 weeks cloning

Comparison of Top Clones from ClonePix System and Limited Dilution

Clone Titer (g/L) qP (pcd)  
Clone 1 4.5 54.6 ClonePix System
Clone 2 4.4 44.6
Clone 3 4.3 49.4
Clone 4 4.0 43.8
Clone A 2.9 32.7 Limiting Dilution
Clone B 2.8 21.0
Clone C 2.7 20.9
Clone D 2.6 29.0
  • Cell lines selected were about 2- fold more productive compared to those selected by conventional approach
  • Sub-clones from same parent (NS/0 cells)

High Titers of Final Clones from ClonePix System Method

Clone Titer (g/L)
1 5.8
2 5.3
3 5.7
4 5.8
  • CHO cells - 14 day fed batch process in shake flasks
  • High titer clones obtained (NS/0: 4-5 g/L, CHO: 5-6 g/L) even prior to process optimization
CONCLUSION FROM MEDIMMUNE: ‘CLONEPIX SYSTEM 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:

Rapid and efficient selection of high producing mammalian cells secreting therapeutic proteins/peptides: Application Note Download

Eliminate Unstable Clones

  • Reveal clonal instability with ClonePix systems
  • 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. 

Example shows results from a selection of stable clones by second round screening System transfers each colony into one well of a 96-well microplate

verify clonal stability, cell line stabilization, clonal screening
Top 2% of transfected population
of suspension- adapted CHO cells
collected and assayed for productivity
selection_of_stable_clones_diagram_2.gif
Productivity vs. fluorescence after second
round screening. Note elimination of
high fluorescence, low productivity
(unstable) clones

Cell confluence and cell number can be tracked by CloneSelect Imager

Antibody Discovery for Research

Identify Higher-Titer Hybridoma Cell Lines For Antibody Development

  • Parental hybridoma cell line was expanded and screened on ClonePix system
  • High yield clones picked by ClonePix System and monitored for growth on CloneSelect Imager System
  • Top growers screened for antigen specificity by target peptide binding assay
  • High valued, specific sub-clones were isolated by pilot production and cell banking
  • Workflow reduced hybridoma screening time by >50% finding optimal producers secreting IgG at higher titers
high-titer-hybridoma.png
White light image
high-titer-hybridoma_FL.png
Fluorescent image

High-throughput screening of hundreds of sub-cloned colonies from parental hybridoma material on ClonePix system aids rescue and stabilization of a high-titer hybridoma cell line secreting highly specific monoclonal antibody to an immunogenic viral antigen.

Further Information

Enhanced Development of Virus-Specific Hybridomas using ClonePix and CloneSelect Imager Technologies: Application Note Download


Imaging Principal - Label-Free Detection of Secreted Antigen-Specific mAbsimage_antigen_0.gif

  • Hybridoma clones grown in semi-solid media
  • Complex Initiation Factor (CIF) traps secreted mAb
  • Fluorescently conjugated antigen targets IgG-secreting clones

Further Information

Screen And Select Antigen-Specific Hybridomas Or B Cells

Screening Antigen-Specific Hybridomas

  • Screen and select antigen-specific clones in situ
  • Suitable for a broad range of antigens
    • - 160kD multimeric protein to 2.6kD phosphopeptide
antigen_pos_w.jpg
White light image
antigen_pos.jpg
FITC conjugated 60kD antigen-specific
IgG producing clone
antigen_neg_w.jpg
White light image
antigen_neg.jpg
FITC conjugated 60kD control protein

Imaging Principal - Label-Free Detection of Secreted Antigen-Specific mAbsimage_antigen_0.gif

  • Hybridoma clones grown in semi-solid media
  • Complex Initiation Factor (CIF) traps secreted mAb
  • Fluorescently conjugated antigen targets IgG-secreting clones

Further Information

Protein Expression and Production

Selection And Development Of GPCR Expressing Cell Lines

Detection and Selection of Cells Expressing GPCR at Low Endogenous Levels

  • Positive (CHO-M1) and negative (CHO-K1) cells plated in CloneMedia and incubated for 8-10 days
  • Labeled antibodies added and cells imaged in brightfield and fluorescent channels
  • Cells visible in both channels picked and cultured
  • GPCR expression validated by fluorescent calcium signals on FLIPR Tetra System
GPCRExpression_l.jpg
System locates and identifies
clones using brightfield
GPCRExpression_r.jpg
Cells positive for GPCR expression
are shown in fluorescent channel

Further Information

  • Rapid Selection and Development of GPCR Expressing Mammalian Cell Lines Using Novel ClonePix Technology: Application Note Download

Select By Intrinsic Reporter Expression

Example: Detection of Intrinsic GFP Reporter 

  • Human breast cancer cells (MCF-7) transfected with internally expressed GFP-fusion protein
  • Cells grown as adherent colonies in MEM Earle’s liquid medium + 10% FBS
  • Day 8: Clones expressing highest GFP selectively picked
intrinsic_white_th.jpg
White light
intrinsic_gfp_th.jpg
GFP
intrinsic_pick_th.jpg
After picking

Select By Cell Surface Expression Marker

  • Adherent CHO K-1 stably transfected with P2Y1 receptorsurface_expression_0.jpg
  • Cells grown in CloneMatrix based semi-solid medium
  • Day 8: anti-P2Y1 receptor polyclonal conjugated with FMAT-Blue added by atomizer
  • Day 9: High expressers of P2Y1 receptor screened and picked

Further Information:

Rapid Automated Selection of Mammalian Cell Colonies by Cell Surface Protein Expression: Application Note Download

Select Secretors Of A Tagged Recombinant Protein

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
tagged_white.jpg
White light image
tagged_fitc.jpg
FITC image

Imaging Principal

image_tag_recomb_1.gif

Stem Cell Screening/Maintenance of Stem Cell Lines

Maintain Lines Utilizing Colony Morphology (White Light)

Maintenance of Human Stem Cells (BG01V hES) Utilizing White Light and Morphology

  • Human stem cells (BG01V hES) grown as colonies on feeder layer in liquid media
  • Plates imaged and colonies picked based on morphology
stem_main_th.jpg
Stem cell clones shown by
white light imaging on
ClonePix System
stem_main_pickedth.jpg
Typical picked clone collected
to 96-well plate. Imaged
on CloneSelect Imager