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.

Summary Many reports have documented apoptotic death in different cell types within hours of exposure to cytotoxic drugs; lower drug concentrations may cause cell cycle arrest at G/M and subsequent death, which has been distinguished from 'classic' apoptosis. We have analysed etoposide-induced cell death in two lymphoblastoid T-cell lines, CCRF-CEM and MOLT-4, specifically in relation to DNA cleavage as indicated by pulse-field gel and conventional electrophoresis. High (5,M) concentration etoposide causes 50-kb cleavage of DNA that occurs at the same time as apoptotic morphology and internucleosomal cleavage. At lower concentrations (0.5-0.05 ,UM), sequential change may be discerned with altered gene expression being similar to that at high dose, but preceding cell cycle arrest and 50-kb cleavage. These last changes, in turn, clearly precede internucleosomal fragmentation of DNA, vital dye staining and morphological evidence cell death. The pattern of higher order fragmentation constitutes a sensitive indicator of commitment to cell death in these cells. Morphological evidence of cell death is associated with internucleosomal fragmentation in one of the lines, but the pattern of 50-kb DNA cleavage provides the clearest evidence of commonality in death processes occurring at low and high drug concentration.

The relationship between apoptosis and cell differentiation has been a subject for continuous debate, with evidence showing leukaemic cell differentiation and drug-induced apoptosis have reciprocal, interdependent and a highly schedule-dependent relationship. We have addressed this relationship in terms of a widely-used model for apoptosis induced by cytotoxic drugs: namely the effect of etoposide on CEM cells. In respect of commitment toward differentiation, we assessed changes in expression of marker genes and the level of CD3 antigenicity. Changes in these parameters following exposure of CEM cells to etoposide was similar to that observed following treatment of the same cells with phorbol 12-myristate 13-acetate (PMA), though this latter treatment did not cause cell death. Similarities in response to etoposide and PMA also included generation of 50 kilobase fragmentation of DNA and convolution of nuclei as assessed by transmission electron microscopy. However, condensation of chromatin and externalization of phosphatidylserine were only recorded in response to the cytotoxic drug and not in response to PMA. The data are consistent with apoptosis in these lymphoblastoid cells being accompanied by activation of specific markers of T-cell differentiation, but ultimately involving processes unequivocally associated with cell death. Cell Death and Differentiation (2000) 7, 548 ± 555.