Mol Biotechnol (2010) 45:257–266 To investigate whether this improvement translated into an increased expression in stable cell lines we did a pool transfection experiment comparing the DG44 and ECHO cell lines. The cells were each transfected with three con- structs encoding different antibodies against Vaccinia virus and transfectants were selected in nucleoside-free medium. Figure 1b shows speciﬁc productivities at the pool level indicating that the ECHO cell line gives productivities that are 3–5 times as high as for the DG44 cell line. A number of antibody-expressing ECHO clones derived by the procedures described here were used for determi- nation of copy number of the integrated construct by Southern blotting and quantitative PCR, and we typically found copy numbers above 100 (not shown). Initial Co-Cultivation of ECHO Clone rpAb Compositions With the aim to investigate whether different stable anti- body-expressing ECHO cell clones generated by means of random integration could be cultivated together to produce an rpAb in a reproducible manner without skewing the com- position or losing diversity, a model study was designed as follows: six different anti-Vaccinia antibodies were selec- ted, each of them having a characteristic cation exchange chromatography (CIEX) proﬁle distinguishable from the others. This facilitated identiﬁcation and quantiﬁcation of the six different antibodies in the compositions. ECHO cells were transfected individually with six different plasmids encoding anti-Vaccinia antibodies and selected for stable expression using the DHFR gene as selection marker. High- producing ECHO clones were isolated by surface labelling of the selected cell pool with ﬂuorescent anti-IgG antibody followed by single-cell sorting by FACS of the 1–2% highest expressing cells into wells containing ECHO cell condi- tioned medium. Twenty four clones were selected for each antibody based on ELISA screening of production levels and visual inspection for growth. These 6 9 24 clones were tested in a 24-well medium exhaustion assay and the 10 highest producing clones for each antibody were adapted to serum-free ProCHO4 medium. Some clones were discarded during adaptation due to poor growth or decreasing pro- ductivity. When adaptation was ﬁnished 5–7 cell clones remained for each antibody. These were all frozen. To test compositional stability of mixed cultures over a long time a number of ECHO clone compositions were prepared that would produce an rpAb consisting of the six different anti-Vaccinia antibodies. Based on cell counting during the late phase of the adaptation period clone doubling times were calculated and taken into consider- ation in the design of compositions to match clones with similar doubling time. Altogether nine compositions were prepared: 261 • • • • • Compositions 1–5: Each antibody was represented by a single clone Composition 6: Two clones were used for each antibody Composition 7: Five clones were used for each antibody Composition 8: Three clones were used for each antibody Composition 9: All available clones were used, 5–7 for each antibody. Clones were mixed so that the number of cells expressing each antibody (for each antibody 1–7 different ECHO clones) constituted 1/6 of the total number of cells in the mixed composition. The idea behind the use of more than one clone for each antibody was that if a clone was lost due to poor growth there were replacement clones present which overall would increase the chance of having all antibodies represented in the ﬁnal mix. The cultivation experiment was performed for a period of 5 weeks. Once a week samples for ELISA and CIEX were taken. The cellular productivity as measured by ELISA was relatively constant over the 5 week period with a tendency toward a slight decline in some compositions (results not shown). The relative content of the six individual antibodies was measured by CIEX at the start and end of cultivation of the nine rpAb cultures (Fig. 2). All antibodies could be detected in all nine cultures both at the start and the end of the experiment. In all cultures, changes in the amount of some of the individual antibodies were seen; in the ﬁnal compositions individual antibodies constituted between 1.7 and 65%. There was not a clear tendency toward less skewing in composition in cultures with more than one ECHO clone representing each antibody (compositions 6–9 in Fig. 2). The number of generations was between 25 and 27. This means that if the cultures had been expanded in full volume every time, the ﬁnal total volumes would be 43,000– 172,000 l. Large scale mammalian cell culture manufac- turing processes in the industry are typically up to 15,000 l. It should however be emphasized that this initial experi- ment did not include cell banks, which means that fewer generations were needed from the ﬁrst mixing of cells to the end point measurement compared to an industrial manufacturing process. Nevertheless, it was encouraging to ﬁnd that the drift seen over this long period of co-culture was not more pronounced. Based on these initial experiments a new set of experi- ments was designed: four new compositions were gener- ated from clones producing the six antibodies replacing cell lines with unwanted behavior (pronounced drift up or