Selective cloning of hybridoma cells for enhanced immunoglobulin production using flow cytometric cell sorting and automated laser nephelometry

Abstract

Techniques for selective cloning of murine hybridoma cells by flow cytometric cell sorting and use of automated laser nephelometry to determine the resultant clones' immunoglobulin secretion levels are described. Using a commercially available attachment to a fluorescenceactivated cell sorter, individual hybridoma cells were successfully distributed into microtiter wells in an automated manner based on their forward angle light scatter properties and their reaction to fluorescein-conjugated anti-mouseIgG. The techniques were used to estimate successfully the frequency of immunoglobulin-secreting cells in established cultures. In addition, heterogeneity of cell surface immunoglobulin expression was observed and utilized as a criterion for flow sorting of new hybridoma variants. In these studies, clones derived from high (anti-IgG) intensity sorting regions yielded cultures with enhanced immunoglobulin secretion levels, as determined by automated laser nephelometry. Furthermore, the surface immunoglobulin phenotype of the derived clones was conserved in subsequent progeny. Finally, it was established that inclusion of propidium iodide in the hybridoma cell sorting mixtures improved cloning efficiency by facilitating enhanced discrimination and elimination of nonviable cells. Our results indicate that flow cytometric-assisted single cell depositioin provides positive attributes of severall traditional hybridoma cloning techniques and, in addition, furnishes a tool for steering the cloning process toward selection of enhanced immunoglobulin producing cultures.

A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics

“…The engagement of surface antibody staining followed by FACSAria cell sorting is the key to obtaining single clones with high antibody productivity. Our results and other reports [2][3][4] suggest that the antibody level transiently displayed on cell surface correlated well with productivity of the cell. Thus, a fluorescein-conjugated antibody can be used to recognize the membrane associated product and aid the isolation of high producers.…”

“…In this operation, production host cells are first transfected with an expression vector carrying the gene of interest 1 , followed by the treatment with a selection agent. The stably-transfected cells are then stained with fluorescence-labeled anti-human IgG antibody, and are subsequently subject to flow cytometry analysis [2][3][4] . Highly productive cells are selected based on fluorescence intensity and are isolated by single-cell sorting on a BD FACSAria.…”

Analysis of an artificially selected GS‐NS0 variant with increased resistance to apoptosis

“…In theory, all cells within these ''clonal'' populations are genetically and phenotypically identical. In practice, however, an entirely homogenous population does not arise, and many industrially important cell lines show a large amount of deviation in growth characteristics of the sub-clones derived from them (Barnes et al, 2006;Kim et al, 1998;Marder et al, 1990). This heterogeneity can allow the selection of cells with advantageous phenotypes either as candidate lines for production or for the analysis of factors contributing to improved phenotypes.…”

Comparative characterization of mAb producing hapten-specific hybridoma cells by flow cytometric analysis and ELISA

“…Several publications have reported some correlation between surface antibody content and antibody secretion rate (Marder et al, 1990;Sen et al, 1990;McKinney et al, 1991;Kromenaker and Srienc, 1994). Sen et al (1990) found a linear correlation between mean surface fluorescence intensity and the specific antibody production rate, while Marder et al (1990) used FACS to isolate clones derived from high fluorescent intensity sorting gates and found enhanced immunoglobulin secretion from these clones.…”

“…Previous flow cytometric studies of hybridoma cells aimed to detect antibodies on the cell surface using only fluorescein-labeled anti-mouse IgG (Marder et al, 1990;Sen et al, 1990;McKinney et al, 1991;Kromenaker and Srienc, 1994;Cherlet et al, 1995;Borth et al, 2000). The lack of information on the specificity of the hybridoma cells, however, is the major disadvantage of this method and therefore it has not been used on a routine basis, yet.…”