Numerous attempts have been made to analyze populations of cells and to separate cells based on the products that they produce. Such approaches to cell analysis and separation are especially useful in assessing those cells which are capable of secreting a desired product (the “product”) or which are relatively high secretors of the product. These methods include cloning in microtiter plates and analysis of the culture supernatant for product; and cloning in agar and analysis by methods for identification of the product of the localized cells; the identification methods including, for example, plaque assays and western blotting. Most methods for analysis and selection of cells based on product secretion involve physically isolating the cell, followed by incubation under conditions that allow product secretion, and screening of the cell locations to detect the cell or cell clones that produce the product. When cells are in suspension, after the cells have secreted the product, the product diffuses from the cell without leaving a marker to allow identification of the cell from which it was secreted. Thus, secretor cells cannot be separated from non-secretor cells with these types of systems.
The affinity matrix technology was established by Manz and coworkers (1995) to study the secretion of antibodies by hybridomas and cytokine secretion of activated T lymphocytes. To create an artificial affinity matrix on the cell surface, primary amine residues of surface molecules were biotinylated. Next, cells were labeled directly with an avidinated catch antibody specific for the secreted molecule. Afterwards, cells were allowed to secrete in gelatin, a medium with decreased permeability to reduce diffusion of the secreted molecule. The catch antibody could then capture the secreted molecules in the vicinity of the cell surface. Secreting cells were subsequently labelled with fluorescent reagents, specific for the secreted molecule, to allow flow cytometric analysis and cell sorting. Borth et al. (Biotechnology and Bioengeneering, 2001, 71: 266-273) used this technique to select for high producing subclones of antibody-secreting CHO cells. CHO cells provide glycosylation patterns of the synthesized proteins that are similar to those provided in human cells. Such proteins are particularly suitable as therapeutic proteins.
A further development of this technique was the use of an (neutr)avidin bridge and a biotinylated antibody instead of an avidinated antibody to maximize the binding capacity of the matrix. Furthermore, the CHO cells were embedded in medium with only 10% gelatin. Holmes and Al-Rubeai (1999) named this method the affinity capture surface display (ACSD).
An alteration of the ACSD method was introduced by Carroll and Al-Rubeai (2005) that replaced fluorescent activated cell sorting by magnetic activated cell sorting to improve cell viability of fragile cells.
In these approaches, the extracellular surface of the CHO cells is chemically treated to allow for a capturing moiety to bind to the extracellular surface of the CHO cells. Moreover, these approaches are laborious and thus disadvantageous.
USPTO Backgrounds
USPTO Backgrounds