Accelerated cell line development using two‐color fluorescence activated cell sorting to select highly expressing antibody‐producing clones

Abstract

The success of engineered monoclonal antibodies as biopharmaceuticals has generated considerable interest in strategies designed to accelerate development of antibody expressing cell lines. Stable mammalian cell lines that express therapeutic antibodies at high levels typically take 6-12 months to develop. Here we describe a novel method to accelerate selection of cells expressing recombinant proteins (e.g., antibodies) using multiparameter fluorescence activated cell sorting (FACS) in association with dual intracellular autofluorescent reporter proteins. The method is co-factor-independent and does not require complex sample preparation. Chinese hamster ovary (CHO) clones expressing high levels of recombinant antibody were selected on the basis of a two-color FACS sorting strategy using heavy and light chain-specific fluorescent reporter proteins. We were able to establish within 12 weeks of transfection cell lines with greater than a 38-fold increase in antibody production when compared to the pool from which they were isolated, following a single round of FACS. The method provides a robust strategy to accelerate selection and characterization of clones and builds a foundation for a predictive model of specific productivity based upon on two-color fluorescence.

Automation of cell line development

“…Several techniques have been developed with the intention to reduce time constraints and manpower costs during cell line development. This includes fluorescence based systems such as fluorescence-activated cell sorting, FACS (Carroll and Al-Rubeai 2004;Brezinsky et al 2003;Sleiman et al 2007), and more recently, the Genetix ClonePix FL system. We have chosen another approach to reduce cost and decrease labour, which is not dependent on fluorescence technology.…”

Generation and Screening of Pichia pastoris Strains with Enhanced Protein Production by Use of Microengraving

“…Furthermore, cells must be incubated in semisolid medium and cannot be screened immediately after cultivation by fermentation. Flow cytometry (FC) has also been used to select both mammalian and microbial strains based on intracellular, surfacebound, or locally encapsulated reporting molecules that link protein production with the expression of fluorescent markers (8,12). These approaches are rapid and relatively inexpensive, but the analysis required to identify and translate beneficial genetic factors into production strains can be costly and require significant effort.…”

Engineered polymer nanoparticles for intracellular delivery

“…Enveloped viruses either fuse to the cell membrane or the endosomal membrane while non-enveloped viruses use specific virion proteins to either penetrate or disrupt the cell or endosomal membranes. 21 No matter what method is used to enter the cell and escape the endosome, once in the cytosol the virus is transported to its normal site of replication, which is most commonly the nucleus. Apart from having high transfection efficiency, other advantages viral vectors have include their ability to protect the transgene by encapsulating it prior to and during internalisation.…”

“…21 The CvME pathway is characterized by flask-shaped membrane invaginations that are lined with caveolin and enriched with cholesterol and sphongolipids. 20b The size of the resulting endocytic vesicle is typically between 50-80 nm.…”

“…from a cationic to anionic polymer), in which degradation is independent of both the molecular weight of the polymer and pH of the environment, nanoparticles. 21 This polymer has also been shown to bind strongly to and release siRNA at a defined time (~17 h), independent of the environmental conditions. 22 The advantage of this polymer is its ability to release where external or environmental triggers are not accessible or can be variable.…”