Improve titer, quality and speed in cell line development.
A robust cell line development platform requires rapid and reliable determination of single-cell origination and subsequent monitoring of clonal cell growth. Traditional techniques, however, using visual microscopic screening to identify and track clonal cell lines in 96-well plates, are challenging, labour-intensive, and highly subjective. In addition, visual identification of single cells in 96-well plates by microscopy is virtually impossible at the scale required to screen hundreds of clones. As such, colonies are usually detected late in development and require second-round cloning to reduce the risk of propagating multi-cellular production lines.
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Figure 1: CHEF1 plasmid for the expression of therapeutic proteins with the regulatory sequences of the CHEF1-gene and DHFR for the selection of positive clones. |
At CMC ICOS Biologics, our core cGMP manufacturing services rely heavily on mammalian cell culture processes, so clonal cell line development is a crucial aspect of our business. The introduction of an automated imaging system (Genetix’ CloneSelect Imager) has vastly improved our ability to accurately assess monoclonality and cell confluence and, in addition, our ability to select rapidly growing, high productivity clones. The system has now become an essential tool in our workflow, saving time and providing evidence of monoclonality that is vital in a regulated environment.
In this article we describe our early cell line development process and platform, including stages at which introduction of the automated imager has assisted our workflow. The cell culture expression platform we use is based on the CHEF1 plasmid (Figure 1), which utilizes the regulatory sequences of the Chinese hamster EF-1a (CHEF1) gene to promote protein production.1 The expression platform allows rapid isolation of stable CHO cell lines, as it can achieve high levels of expression without the need for methotrexate amplification. Early identification of CHEF1 monoclonal cell lines with the CloneSelect Imager further expedites the clone selection process. In addition to establishing monoclonality, we are currently using the imager to monitor and correlate clonal cell growth with later-stage productivity in batch production models.
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Figure 2: CloneSelect Imager colorized plate thumbnail image illustrating typical cell growth in a 96-well cloning plate. |
Methods
A model antibody was cloned into the CHEF1 plasmid with the DHFR gene as a selection marker. The cell line CHO DG44 (DHFR-) was then transfected with the resulting plasmid by standard electroporation, with three day recovery in non-selective media. Following recovery, the cells were grown for two weeks in our proprietary serum-free, chemically defined selection medium lacking hypoxanthine and thymidine. Those cells that are not successfully transfected with the CHEF1 plasmid, and therefore lack the DHFR gene, die during selection. The viability of the cell culture decreases to approximately 40% and then typically recovers to over 90% at the end of the two week period.
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Figure 3 (top): Clonal images taken from single cell to colony stage on Day 14 without mechanical disruption, using CloneSelect Imager.
Figure 4 (bottom): Non-clonal images taken from two cells that grew into a single colony on Day 14 without mechanical disruption, using CloneSelect Imager.
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As soon as transfected cell pools reach greater than 90% viability they are diluted to an estimated seeding density of one cell per well in serum–free cloning media, in 96 well plates, and grown for two weeks. The first images are taken with the CloneSelect Imager on Day 0, approximately four hours post-seeding in order to give the cells time to settle. Subsequent images are taken at Days 1, 3, 7 and 10. By Day 10, well formed colonies exist and at this point monoclonal colonies are identified by the CloneSelect Imager and verified by manually viewing the Day 0, 1, 3, and 7 images. Confirmed monoclonal cell lines are then simultaneously fed fresh media and disturbed
in situ by mechanical pipetting. This action disburses the cells in the wells, after which they are allowed to grow for three to five days before imaging again with the CloneSelect Imager to measure changes in percent confluence (cell density).
Results and Discussion
The CloneSelect Imager is used to track cell growth in 96-well plates throughout a two week clone selection period. Previously, we would have needed to establish monoclonality through the use of FACS single cell seeding, bracketed seeding densities, or multiple dilution cloning rounds, all of which are very time consuming. Using the CloneSelect Imager, we can accurately track and record growth in each well, thereby determining individual growth. The ‘plate thumbnail’ functionality highlights colonies with green color allowing rapid screening of 96-well plates to determine which wells contain colonies (Figure 2), and the ‘loci of growth’ functionality allows us to track cells at each time point throughout the growth period (e.g. days 1, 3, 7, and 14), thereby facilitating verification of colony origin by tracking the image history of each well (Figure 3). Reviewing the archival Day 0 or Day 1 images is critical as what may appear to be a single colony on Day 7, or later, may have actually derived from two cells (Figure 4).
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Figure 5 (top): Microscopic (white light) and color enhanced images of a Day 10 colony compared to subsequent Day 11, 13 and 15 images after mechanical disruption and feeding.
Figure 6 (bottom): Percent confluence (cell density) for 48 clonal cell lines in 96-well plates was measured and ranked from high to low based on the Day 13 values.
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Recently, we have used the CloneSelect Imager to measure changes in colony growth to rapidly identify cell lines with desirable growth characteristics. To promote growth, colonies were fed fresh media and mechanically dispersed on Day 10 and then imaged on Days 11, 13 and 15. Using the imager we tracked colony outgrowth and utilized the ‘confluence’ functionality to measure the density (area) of cells in an individual well for 48 confirmed monoclonal cell lines over three days (see example in Figure 5). The percent confluence was plotted for each clone (Figure 6) to illustrate the range of growth profiles that, if arbitrarily split into four groups of twelve, delineates clones with slow growth on Day 11 to 13 (Clones 1 to 12) from those with fast growth (Clones 37-48). By Day 13, most of the fast growing cells reached near-maximum density (100% of well) and slowed in growth, whereas the slow growers continued to grow from Day 13 to 15.
By tracking colony outgrowth it is possible to create initial growth profiles for clones in 96-well plates that can then be correlated to protein productivity in larger tissue culture vessels. The 48 clonal cell lines were expanded into 50 milliliter spin tube production models (10 ml working volume) and run in batch mode for six days. The average productivity (titer) of the 48 clones split into four groups of twelve, Clones 1-12, 13-24, 25-36 and 37-48, is shown in Figure 7. There is a trend towards increased protein expression in the fast growing clones (Clones 37-48) compared to those with lower Day 13 growth. The fast growing clones (Clones 25-36 and 37-48) had slightly higher viable cell densities in the production models, probably contributing to the higher titers and suggesting early growth characteristics persist after expansion into suspension growth. These data indicate that early stage clonal growth measured with the CloneSelect Imager may be a useful predictor of future expression potential and can be used as a preliminary screen to reduce the number of clones taken forward. Thereby, in our standard cell line development platform we are able to use the CloneSelect Imager both to establish monoclonality and to limit the number of forward processed clones. These developments have improved our throughput and clone selection process to yield production-quality clones in just 10 weeks from transfection.
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Figure 7: Average titers (mg/L) and viable cell densities (VCD x 105 cell/ml) for groups of 12 clonal cell lines, based on Day 13 confluence, in 6-day batch production model.
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Summary Using the workflow described above, we have been successful in generating high quality production clones, which provide good growth and high productivity, in a very rapid time frame. Rapid selection is made possible based on both the robustness of the CHEF1 plasmid and cell lines, and secondly, the predictable growth behaviour of the clones that are selected using our screening platform. Using the CloneSelect Imager system gives us confidence in the clonal origin and predicted growth characteristics of the selected clones, and we are thus able to reduce the number of clones screened to find a suitable production-quality cell line.
References
1. Jennifer Running Deer and Daniel S. Allison. High-Level Expression of Proteins in Mammalian Cells Using Transcription Regulatory Sequences from the Chinese Hamster EF-1r Gene. Biotechnol. Prog. 2004, 20, 880-88