CellXpress™ powered by LEAP accelerated development of highly-secreting cell lines

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The generation of stable cell lines with high protein secretion is challenging and represents a major bottleneck in biopharmaceutical process development. The inefficiencies of current methods for cell line selection and generation have created an urgent need for innovative approaches that shorten timelines and improve productivity. This application note presents a novel approach to cell line generation and characterization, based on in situ measurement of specific protein secretion followed by in situ cloning, which meets this critical need.

Cell cloning for therapeutic protein production is typically performed by limiting dilution, an inefficient and random process that is not amenable to automation, leading to cloning of many non-secreting cells in addition to the desired secreting cells. Significant maintenance and growth of all clones is required before protein secretion can be measured (e.g., by HPLC), and the process usually requires a significant number of long, serial sub-cloning steps (Underwood and Bean 1988).

CellXpress Benefits
From pool to highly-secreting clones in less than 24 hours
Ten-fold more highly-secreting clones in less time
Select optimal clones for secretion, growth and stability
Less than half the time to validated clones
Up to 90% reduction in labor and material costs
Real-time growth rate tracking
Process multiple projects / cell lines in parallel

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Fig. 1. (Transfected Pool) Pool of heterogeneous IgG secreting suspension-adapted CHO cells. Cells are identified by green fluorescence. Cell-associated IgG secretion halos are identified by red fluorescence. (CellXpress Clone) The single remaining highest secreting cell after CellXpress-mediated in situ clonal purification. (Expanded Clone) Homogeneous cell line population after outgrowth of CellXpress clone for ~50 doublings.

The cost of limiting dilution is considerable in terms of time, personnel, and materials, generally requiring 9-18 weeks to reach the first meaningful secretion validation checkpoint. This checkpoint comes only after managing large numbers of unvalidated clones for an extended period of time. Further, the selection pool size is generally limited to <1,000 cells. CellXpress™ is a fundamentally new approach which combines quantiÂ¬tative in situ secretion assessment of individual living cells with laser-mediated elimination of all non-and poorly-secreting cells, leaving only the highest-secreting cell in every well. Recombinant cells secreting protein are cultured on a solid capture matrix, followed by staining with a fluorescent detection reagent (Hanania et al. 2005). The Laser-Enabled Analysis and Processing (LEAP™) system images and locates every cell within a well, quantifying the cell-associated and secreted protein, and then eliminates all undesired cells from a well via targeted laser irradiation. Clones are verified and clonal outgrowth followed during expansion using brightfield imaging, providing very early measurement of clonal growth rates. The ability to assay cell secretion on single cells further enables measurement of the distribution of secretion within the cell population, allowing assessment of secretion stability well in adÂ¬vance of having sufficient cells for conventional assays. CellXpress has been successfully applied to multiple cell types including commonly used CHO, NS0, and hybridoma cells.

Results

Recombinant CHO cells producing humanized antibody were assayed using the CellXpress Kit. Cells were cultured in normal growth medium on a proprietary capture surface in a 384 well C-lect™ plate. The next day, secreting cells were identified by staining with a viable cell dye and a human antibody-specific detection reagent. The CellXpress application running on LEAP was used to identify and clone the single highest secreting cell in each well by eliminating all other cells in each well (Fig. 1). CellXpress is quantitative, providing a means of directly selecting cells for cloning based on specific productivity (Fig. 2).

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Fig. 2. Quantitative comparison of CellXpress secretion measurement and HPLC. Several clonal cell lines secreting differing levels of IgG were analyzed by both CellXpress and conventional HPLC. In situ secretion measured by CellXpress was highly correlated with HPLC measurement from supernatant.

Plates (384 well) containing ~200 antibody-secreting CHO cells per well were processed in <1 hr per plate. After a single round of laser targeting, over 30% of the wells contained a single secreting clone. This number increased to over 60% if a second processing iteration was included on the same day. The cells were processed in situ with the plate lid in place, making this a closed system process. Single highly-secreting clones isolated by this method resulted in expansion to cell lines with superior homogeneous secretion characteristics (Fig. 1).

An additional unique feature of CellXpress is the ability to track clonal outgrowth from a single cell using brightfield imaging (Fig. 3). This feature facilitates submission of production cell lines to regulatory agencies, providing a complete history of each cell line from initial purification to a single cell and all subsequent clone outgrowth steps.

The ability to assay each living cell for secretion provides an opportunity to reassess specific productivity on a very small cell sample at each stage of passage during expansion. Clones that have reduced or stopped secreting the protein product (Fig. 4) can be identified and removed, thereby reducing the overall effort. Identification of unstable clones can be observed as early as 2 weeks before there are sufficient cells to perform conventional assays.

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Fig. 3. Brightfield Confirmation of Clonal Outgrowth. Brightfield imaging was used after CellXpress-mediated cloning to confirm clonality at the 1 cell (immediate) and the 1 colony (1 week) stage.

Additional CellXpress Benefits
Image-based validation of clonality
Process cells directly in normal growth medium
Process cells in sterile, closed plate environment
Higher throughput / scalable / automatable
Clone from pools up to 600,000 cells in 1 day

CellXpress has provided 10-fold more highly-secreting clones from a given selection pool with only ~4-8 weeks to the first secretion validation checkpoint (HPLC assay). Processing of a plate is automated, dramatically reducing personnel time compared to limiting dilution. A single 384 well plate provides selection of the top ~0.25% secreting cells from a selection pool of >75,000 cells.

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Fig. 4. Assay of secretion heterogeneity/stability enables reduction in workload. Assessment of the distribution of secretion levels within populations of cells provides insight into the quality of the culture. As early as Day 30, this clone exhibited an increase in non-producers within the population (lower population), even while the mean production level was maintained (red bar). This increase in non-producers was predictive of the future loss of overall production.

Conclusion

CellXpress is a powerful application for rapid and robust generation of highly-secreting biopharmaceutical cell lines. The unique nature of the application guarantees isolation of a single cell clone and enables detailed tracking of colony expansion and secretion. The quantitative image analysis associated with CellXpress provides accurate assessment of clone secretion in the earliest stages, thereby eliminating effort wasted on non-productive clones. The iin situ nature of the process also allows serial enrichment steps prior to cloning, further maximizing robustness of resulting clones. CellXpress has proven to significantly reduce the time and effort in the generation of high value cells for biopharmaceutical manufacturing.