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Feb 15, 2008 (Vol. 28, No. 4)

Automating Clone Picking in Bioproduction

Genetix Combines a Myriad of Tools and Techniques to Reduce Labor

  • Selection of high-productivity cells is currently a major bottleneck in the manufacture of recombinant proteins and mAbs. Genetix has developed a system, ClonePix FL, that brings together biology and advanced imaging with integrated robotics and data tracking to automatically pick the highest value clones in a single step. The technology allows the high producers to be selected at a much earlier stage of the biopharmaceutical value chain and eliminates much of the manual labor required by traditional clone selection methods, according to the company. 

    Genetix started out in 1991 by developing robotics and plasticware. It was involved in the human and rice genome projects, contributing technology for high-throughput bacterial colony picking and then, in 2003, it launched a mammalian cell picker.

    “We found other niches for our automated systems and evolved from genomics to proteomics applications,” explains Hilary Latham, marketing manager. Accordingly, the company then moved into developing software and systems for phage display, protein-protein interaction, enzyme evolution, and clone management—adding value to what was traditionally done by hand, Latham reports.

    ‘“It became clear to us that the growth in the market would be in production of recombinant proteins, especially antibodies,” says scientific director Julian Burke, Ph.D. “We developed the biology to identify high-producing mammalian cells for production. We select the best clones early on in production so that significantly less tissue culture on cell lines is needed.”

    Screening cell populations for high-producing clones traditionally involves cell sorting, limiting dilution, ring cloning, or even simple manual collection of colonies. All are time consuming, labor intensive, costly, and prone to cross-contamination and error. That is why cell line selection is a major bottleneck in biopharmaceutical production. 

    “You are limited in what you can do manually,” says Latham. “Finding a high-producing clone is like finding a needle in a haystack. ClonePix FL is unique because it can screen far more clones than you could do manually. It enables you to find the needles in the haystack that you would not be able to discover manually.”

    How It Works

    Details of the setup vary with application but, in the simplest scenario, the system uses a target-protein-specific capture antibody conjugated to a fluorophore to probe colonies of producing cells. The imaging system software selects high-producing clones, typically the top one or two percent, and directs the robot to pick them off into microwell plates for further processing and analysis. 

    The workflow of cell selection with the ClonePix FL begins with transfection of the cell line and growing it with standard methods prior to plating into six-well plates with Genetix’ semisolid medium, CloneMedia or CloneMatrix, depending on application. The former is a complete medium for CHO or hybridomas, the latter is a concentrate to which customized medium can be added. In either case, the semisolid nature of the medium is important; it immobilizes the cells while they grow, divide, and form colonies and traps the complex that forms between the fluorescent capture probe and the secreted protein in the vicinity of the secreting clones, according to the company.

    The fluorescently conjugated capture and detection antibodies are either added to the medium at the time of plating or they can be sprayed on with an atomizer 24 hours before imaging and picking the colonies. 

    The system is compatible for both suspension adapted cells and adherent cells. The latter, like CHO-K1 and HEK 293, can be grown as either adherent colonies in tissue culture-treated plates or as suspension colonies in nontissue culture-treated plates. The PetriWell six-well and one-well plates used for the process have been designed specially for the application to minimize the autofluorescence and flaring found with regular culture plates. The plates have an integrated water reservoir to retain the high humidity necessary for optimal growth of cells in semisolid medium.

    After the colonies have grown to about 100 cells, typically 7–10 days, they are imaged. The ClonePix FL system captures both white light and fluorescent images in situ to quantify the clones and their protein-secretion levels. Up to five different target proteins can be probed by multiplexing different fluorophores in the same reaction. Clones with the required characteristics are selected automatically and placed in order of preference into 96-well plates.

    The picking is carried out with a multichannel picking head with eight individually addressable picking pins. The colonies are picked up gently, retaining cell viability, and placed into 96-well plates for assay and expansion. The colonies can be dispensed intact or mechanically dispersed. The software allows tracking of each colony from its source-plate position to the microwell in its destination plate.

    ClonePix FL has a low labor requirement. It can screen thousands of clones and selectively collect the highest value clones in just a couple of hours. It is estimated that one ClonePix FL used by two scientists will be enough to take more than 100 cell populations from transfection to the shake-flask stage each year. Therefore, the amount of labor required to develop a cell line can be reduced by as much as 10-fold, Latham says.

    ClonePix FL has a range of applications. For instance, it can overcome traditional inefficiencies in screening hybridoma fusions for antigen-specific clones, reports the company. The ClonePix FL system identifies and collects only those cells secreting antibodies that specifically recognize the antigen. They are probed in situ with antigen and IgG-specific Complex Initiation Factor, which allows selection of antigen-specific, IgG-secreting clones. The system works with a range of antigens from 1 kD synthetic peptides to 160 kD multimeric proteins.

    Also of use in therapeutic antibody production is the ability of ClonePix FL to identify the highest-productivity cells. Here, the more cells that can be screened the better, while the current method using limiting dilutions is somewhat empirical and involves many tissue culture manipulations. ClonePix FL probes transfected CHO cells and myelomas with an in situ fluorescence assay to detect and measure levels of IgG secretion. Once the clones have been isolated, the system is also used to test their stability. An aliquot of cells is replated and level of fluorescence checked over a period of time to be sure it is consistently high.  

    The system has also been used to identify and isolate cells producing recombinant proteins other than monoclonals. In the special case of monomeric-secreted proteins, which, by their nature, cannot form a secretion complex, applications have been developed to detect the best secretors by probing epitope tags constructed onto the recombinant protein. 

    Another application is the selection of stable cell lines for screening assays. The system selects clones based on direct fluorescence from reporter proteins like GFP.  The fluorescence intensity indicates clones with stable protein expression or correct protein conformation, which will be the most suitable in a cell-based assay.

    Finally, ClonePix FL is also gaining some interest from users in stem cell applications because of its ability to distinguish between differentiated and pluripotent cells. Plates are imaged with white light, and the relevant clones are detected by fluorescent markers of pluripotency such as anti-SSEA-1.

    Potential

    In the future, Genetix wants to extend its capabilities along the biopharmaceutical value chain. To this end, it bought Applied Imaging in November 2006, which gives it access to two new imaging platforms, CytoVision and Ariol.
    CytoVision allows imaging of chromosomes in applications such as leukemia diagnostics. Genetix has built a new automatic slide loader and oiler for the system. Meanwhile, Ariol is a platform for tissue analysis that can be used, for instance, to analyse breast tissue for expression of HER2, which indicates the patients that could benefit from treatment with Herceptin. It can also be applied to target discovery.

    “By buying Applied Imaging, we have acquired systems that can image cells for applications from target identification to clinical diagnosis,” says Dr. Burke. “It fits with our mission to deliver technologies to our customers that have a competitive edge in drug discovery.”



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