Mitochondrial membrane potential identifies cells with high recombinant protein productivity.

Development of cell lines for biotherapeutic protein production requires screening large numbers of clones to identify and isolate high producing ones. As such, stable cell line generation is a time- and resource-intensive process. There is an increasing need to enhance the selection efficiency of high-yielding clonal cell lines for cell line development projects by using high throughput screening of live cells for markers predictive of productivity. Single cell deposition by fluorescence activated cell sorting (FACS) is a commonly performed method for cloning to generate cell lines derived from a single recombinant cell. We have developed a novel strategy to identify higher productivity cells at the FACS step by leveraging a simple viable cell staining method that detects mitochondrial membrane potential (Ψm), a key indicator of cellular metabolic activity. We chose a dual-emission dye (Mito-ID, Enzo Life Sciences) that fluoresces green and orange in living cells with the intensity of the orange fluorescence being dependent on the cells Ψm status. Using available clonal cell lines with known productivity, or stable transfectant pools, we evaluated Ψm of cell populations with Mito-ID dye. We determined that the intensity of the Ψm fluorescent signal correlates with the known fed-batch titers of the producer clones, and that cell sorting based on an optimal Ψm staining intensity selectively enriches for higher producing clones from nonclonal transfectant pools. These clones are phenotypically stable for recombinant protein production. Furthermore, the strategy has been successfully applied to identifying higher producing cell lines for a range of antibody molecular formats. Using this method, we can combine an enriching step with the cloning step for high producers, thereby saving time and resources in cell line development.

High-throughput screening of antibody-expressing CHO clones using an automated shaken deep-well system.

Biopharmaceutical protein manufacturing requires the highest producing cell lines to satisfy current multiple grams per liter requirements. Screening more clones increases the probability of identifying the high producers within the pool of available transfectant candidate cell lines. For the predominant industry mammalian host cell line, Chinese hamster ovary (CHO), traditional static-batch culture screening does not correlate with the suspension fed-batch culture used in manufacturing, and thus has little predictive utility. Small scale fed-batch screens in suspension culture correlate better with bioreactor processes but a limited number of clones can be screened manually. Scaled-down systems, such as shaken deep well plates, combined with automated liquid handling, offer a way for a limited number of scientists to screen many clones. A statistical analysis determined that 384 is the optimal number of clones to screen, with a 99% probability that six clones in the 95th percentile for productivity are included in the screen. To screen 384 clones efficiently by the predictive method of suspension fed-batch, the authors developed a shaken deep-well plate culturing platform, with an automated liquid handling system integrating cell counting and protein titering instruments. Critical factors allowing deep-well suspension culture to correlate with shake flask culture were agitation speed and culture volume. Using our automated system, one scientist can screen five times more clones than by manual fed-batch shake-flask or shaken culture tube screens and can identify cell lines for some therapeutic protein projects with production levels greater than 6 g/L. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1460-1471, 2018.

Measuring the aggregation of CHO cells prior to single cell cloning allows a more accurate determination of the probability of clonality.

The manufacturing process for biotherapeutics is closely regulated by the Food and Drug Administration (FDA), European Medicines Agency (EMA) and other regulatory agencies worldwide. To ensure consistency of the product of a manufacturing cell line, International Committee on Harmonization guidelines (Q5D, 1997) state that the cell substrate should be derived from a single cell progenitor, i.e., clonal.Cell lines in suspension culture may naturally revert to cell adhesion in the form of doublets, triplets and higher order structures of clustered cells. We can show evidence of a single colony from limiting dilution cloning or in semi-solid media, but we cannot determine the number of cells from which the colony originated. To address this, we have used the ViCELL® XR (Beckman Coulter, High Wycombe, UK) cell viability analyzer to determine the proportion of clusters of two or more cells in a sample of the cell suspension immediately prior to cloning. Here, we show data to define the accuracy of the ViCELL for characterizing a cell suspension and summarize the statistical model combining two or more rounds of cloning to derive the probability of clonality. The resulting statistical model is applied to cloning in semi-solid medium, but could equally be applied to a limiting dilution cloning process. We also describe approaches to reduce cell clusters to generate a cell line with a high probability of clonality from a CHO host lineage. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:593-601, 2018.

Transfection of cultured eukaryotic cells using cationic lipid reagents.

The development of high-efficiency methods for the introduction of functional genetic material into eukaryotic cells using cationic lipids has accelerated biological research in the studies of gene expression, control of cell growth, and cell lineage. Transfection mediated by cationic lipids is commonly used in industrial protein production as well as in some clinical gene therapy protocols. This unit describes how to perform transfection of adherent and suspension cells, insect cells, and RNA transfection using cationic lipid reagents.

Transfection of cultured eukaryotic cells using cationic lipid reagents.

The development of high-efficiency methods for the introduction of functional genetic material into eukaryotic cells using cationic lipids has accelerated biological research in the studies of gene expression, control of cell growth, and cell lineage. Transfection mediated by cationic lipids is commonly used in industrial protein production as well as in some clinical gene therapy protocols. Replacing our previous unit on this topic, this new version describes how to perform transfection of adherent and suspension cells, insect cells, and RNA transfection using the cationic lipid system.