262 Fig. 2 Relative content of six anti-Vaccinia antibodies in nine compositions maintained in co-culture in 50 ml culture tubes for 6 weeks. The nine compositions containing six different anti-Vaccinia antibodies (I–VI) with one or more clonal ECHO cell line(s) expressing each antibody were prepared as described in ‘‘Materials and Methods’’ section. Supernatants were analyzed for content of the individual antibodies by CIEX at the start (dark gray columns) and end (light gray columns) of the cultures. The relative content of the six antibodies (along the horizontal axis) is given as percentage of total antibody (vertical axis) Mol Biotechnol (2010) 45:257–266 down) with cell lines with desired growth characteristics. The compositions were 1A, 2A, 3A, and 4A of which 1A–3A contained one ECHO clone per antibody and 4A contained three ECHO clones per antibody. To better mimic a real manufacturing situation, polyclonal master and working cell banks (pMCBs and pWCBs) were pre- pared and a production phase was performed in bioreactors. The results showed that by removing clones with unwanted characteristics, the new compositions showed a less pro- nounced skewing of the antibody composition during the culture period. Furthermore, when the compositions were produced in duplicate using separate parallel production runs and starting seed-train expansions by thawing of dif- ferent pWCB ampoules a high degree of batch-to-batch consistency resulted for all four compositions (Fig. 3). Overall, these ﬁrst experiments were promising regard- ing the possibilities for producing rpAb in a reproducible manner using an iterative process for the selection of an appropriate composition of clones. The following experi- ments were initiated to conﬁrm its usefulness using a dif- ferent viral target. Preparation of Anti-Respiratory Syncytial Virus rpAb Compositions Next, the feasibility of the SympressTM II system was tested using a completely different rpAb composition, consisting of six antibodies all targeting the Respiratory Syncytial virus (RSV). For each of the six antibodies at least six different stable ECHO clones were generated and banked individually. Compositional stability studies with different combinations of the anti-RSV antibody-express- ing ECHO clones were performed in suspension culture in chemically deﬁned medium. In order to mimic a large scale industrial manufacturing process studies were performed according to the scheme in Fig. 4. Different rpAb combinations of the anti-RSV ECHO clones were mixed and banked as pMCBs, followed by preparation of pWCBs. pWCB ampoules were thawed, seed-train expansion was performed in shaker ﬂasks, and a production run was performed in 500 ml DASGIP biore- actors using a fed batch process as typically used also for mAb manufacturing. The studies were designed to simulate all the steps included in an industrial manufacturing pro- cess at 5,000–10,000 l scale as closely as possible. Thus, before banking of the pWCB as well as during seed-train expansion before inoculation of the bioreactor the cells were cultivated for the number of generations required to perform these steps in a scaled-up industrial process. The relative amounts of the individual antibodies in the rpAb compositions were analyzed by CIEX at the end of the experiment. An example of a CIEX proﬁle of an anti-RSV composition is shown in Fig. 5. The single anti-RSV antibodies elute in distinct peaks and can be individually quantiﬁed by integration of the peak areas. In an exploratory ﬁrst set of compositional stability studies different compositions were prepared by combining clones with as similar doubling times as possible. Apart