264 Mol Biotechnol (2010) 45:257–266 Fig. 5 CIEX proﬁle of an anti-RSV rpAb composition containing six different anti-RSV speciﬁc antibodies (Ab1–Ab6). The borders of the six individual peaks are marked by the vertical lines. Integration of the area of the individual peaks is used for relative quantiﬁcation of the antibodies in the rpAb Fig. 6 Relative concentrations of the six different anti-RSV antibod- ies after single-batch manufacturing in fed batch mode. Three different combinations of stable ECHO clones expressing the anti- RSV rpAb were prepared and tested for compositional stability in a fed batch setup at 500 ml scale designed to simulate production at 5,000–10,000 l scale. The supernatant harvested after single-batch manufacturing was puriﬁed by protein A capture and the antibody compositions were analyzed by CIEX. The CIEX peaks were quantiﬁed by integration to obtain the relative distribution of the individual antibodies in the compositions. a Composition I, b composition II, c composition III. All runs were performed in duplicate Optimized compositions were generated by replacing clones which had contributed to compositional instability by either taking over or disappearing from the culture with other clones showing better characteristics. Seed- trains and production runs were performed in duplicate for each composition after thawing of two different pWCB ampoules and following a seed-train and fed batch pro- duction scheme mimicking an industrial process at 5,000– 10,000 l, as described above and in Fig. 4. In this set of experiments, several compositions were generated with an acceptable distribution of the individual antibodies after the production run. Also, the reproducibility of the parallel seed-trains and production runs was high (Fig. 6a, b). In a third round of compositional stability studies the information gained on the different ECHO clones in the two previous studies was used by combining clones with matching growth properties and productivities. The experiment was designed essentially as before. In this study compositions were generated with a more equal distribution of the antibodies after the production run (Fig. 6c). Here, the relative concentration of the single antibodies was between 12 and 25% at the end of the production run. In order to conﬁrm the batch-to-batch consistency we repeated seed-train expansion and fed batch production of composition I (Fig. 6a). The polyclonal seed-trains were run in four parallel shaker ﬂasks and DASGIP bioreactors. The cells used for the four runs originated from four sep- arately thawed ampoules of the pWCBs. Fed batch runs were as described before and CIEX proﬁles of supernatants were analyzed at day 10 of the fed batch process. As Fig. 7 indicates, the batch-to-batch variation within the four par- allel runs was low. Also, the relative individual antibody concentrations in the supernatant harvested from these consistency runs were similar to the antibody concentra- tions obtained in the previous study. Slight differences might be explained by the different timing of sample taking (10 days vs. 12 days).