Mol Biotechnol (2010) 45:257–266 265 Fig. 7 Comparison of the relative area of the individual anti-RSV antibodies from two independent sets of fed batch production runs performed with rpAb composition I. The dark bars represent the average antibody concentration of four parallel consistency runs. The light bars represent the average antibody concentration of the duplicate runs initially performed with composition I (see Fig. 6a). Samples for CIEX proﬁles were taken at day 10 (right bars) and day 12 (left bars). The standard deviation is indicated as error bars Discussion Here we describe the production of target-speciﬁc rpAb compositions exempliﬁed by two completely different anti- body compositions, namely an anti-Vaccinia virus rpAb and an anti-RSV rpAb, each comprised of six different IgG1 kappa antibodies speciﬁc for their intended target, using stable transfection of cells by random integration and pro- duction in a single-batch format. Surprisingly, the concept of using random integration turned out to be successful in both cases, which makes us suggest that the concept may be generally applicable for rpAb manufacturing. In bioreactor experiments simulating a 5,000–10,000 l production using extended seed-trains, the relative antibody compositions were very similar in parallel production runs. This indicates that batch-to-batch reproducibility, which is mandatory for regulatory approval of rpAbs as human therapeutics, is achieved by this manufacturing strategy. Furthermore, extrapolating the laboratory scale result to industrial manu- facturing scale, the SympressTM II platform provides sufﬁ- cient yields to support commercially sound cost-of-goods. Along with the SympressTM expression platform develop- ment a release and characterization strategy for regulatory approval of rpAb for clinical use have been established and several methods addressing the compositional variability of rpAb products have been established [3, Persson, P., Development of mass spectrometry based methods for identiﬁcation and determination of compositional variability in recombinant polyclonal antibody products, unpublished results]. Having established a consistent rpAb batch-to-batch reproducibility, we wanted to explore to which extent the relative content of the antibodies in a composition could be controlled. For most indications one would envision that a 1:1:1:… antibody distribution would be beneﬁcial, while other antibody ratios could be optimal for certain indica- tions. Thus, with the goal to achieve a relatively equal distribution of the six antibodies in the ﬁnal RSV rpAb composition an iterative approach where clones were mixed based on growth characteristics and productivity was shown to work. The data from the ﬁrst compositions were used to exclude clones that behaved in an undesired manner, showing differential growth compared to the other clones or, alternatively, changing productivity over time. In new experiments it was possible to achieve compositions with a relatively equal distribution of the six antibodies after completion of the production phase. It is well-known that CHO cell clones generated by random integration show a high variability in growth rate and productivity [15]. The production level and the speciﬁc protein to be expressed are also known to affect the clonal growth rate [16]. Polyclonal clone compositions will in an industrial setup have to go through at least 35 cell divisions in order to include two-tiered cell banking and seed-train expansion before reaching the ﬁnal production phase. It was to be expected that cultivation for multiple generations of rpAb compositions expressing different antibodies at slightly different levels with potentially different growth rates would lead to one or more clones taking over the cultures and/or to clones disappearing. As we show here this is certainly seen to some extent, but surprisingly, a few iterations and exchange of clones with undesired charac- teristics made it possible to establish compositions where the rpAb composition is maintained in the ﬁnal product. Importantly, the conceptual studies were designed to sim- ulate a process appropriate for industrial manufacturing at large scale. The applied process includes a two-tiered cell banking step, which is important for a biological product for human therapeutic use in order to secure homogenous seed material for manufacturing throughout product lifespan. Different alternatives could be thought of regarding production of rpAbs. First, all antibodies in a lead com- position could be produced, puriﬁed, and characterized separately before being mixed in the ﬁnal drug product. However, development costs would be high and most likely prohibitive if more than a few clones (3–4) should be present in the ﬁnal composition. An alternative approach could be the use of partly separate culturing of the clones constituting the rpAb compositions: Monoclonal Master and Working cell banks (MCB and WCB) are prepared for the different clones constituting the ﬁnal composition, and each clone is thawed and cultured individually during the whole seed-train or part of it. This would lead to a higher degree of control over the ﬁnal composition since the number of generations in co-culture would be lower,