Mol Biotechnol (2010) 45:257–266 Fig. 3 Relative content of six anti-Vaccinia antibodies from two independent fed batch runs of each of the rpAb compositions 1A, 2A, 3A, and 4A, performed in DASGIP bioreactors at 500 ml scale as described in ‘‘Materials and Methods’’ section. The compositions 1A–3A contained one ECHO clone per antibody and 4A contained three ECHO clones per antibody. Samples for CIEX proﬁle analysis were taken on day 16 of the bioreactor run 263 Fig. 4 Experimental setup of the compositional stability studies with compositions expressing the anti-RSV rpAb. Suspension ECHO cell clones expressing each of the six monoclonal anti-RSV antibodies were banked individually. Selected ECHO clones, one for each of the six anti-RSV antibodies, were thawed and mixed. The mixed compositions were immediately frozen as polyclonal master cell banks (pMCB). One ampoule of the pMCB was subsequently thawed, expanded, and frozen as polyclonal working cell bank (pWCB). pWCB cells were then thawed and seed-train expanded prior to a fed batch production run in bioreactors from testing compositions with one ECHO clone per antibody, we also tested a composition in which each antibody was represented by three different clones. The compositions were frozen as pMCBs, thawed again and expanded for an appropriate number of generations ([8) before preparation of pWCBs. Two pWCB ampoules were thawed for each composition to start parallel seed-trains. This was done in order to examine reproducibility of the production runs. Seed-train expansion was performed in shaker ﬂasks. The relative antibody composition in the duplicate production runs was monitored by CIEX and the reproducibility of the duplicate runs was found to be acceptable. However, in all compositions a tendency toward some antibodies dominating the cultures and other antibodies decreasing in quantity was observed (data not shown). The composition consisting of three ECHO clones per antibody resulted in a highly skewed composition after fed batch production. Based on these results, and because rpAb compositions prepared with one clone representing each antibody provides a simpler approach with respect to cell line characterization, it was decided to concentrate on one-clone-per-antibody compositions. The observa- tions from the exploratory compositional study were used for preparation of new modiﬁed clone compositions (see below).