258 these methods for consistent production of rpAb without substantially increasing the costs of production and regu- latory approval. Target-speciﬁc rpAb is a new generation of antibodies mimicking the diversity, speciﬁcity, and binding capability of the natural human immune system. To do this an rpAb preparation will often contain several speciﬁc antibodies, and for economic reasons a manufacturing procedure with all antibodies being produced in the same culture vessel will be preferable. Obviously, the ability to maintain the same antibody composition between batches, the so-called batch-to-batch consistency, is crucial for getting rpAb approved for human therapeutic use. Maintaining batch-to- batch consistency is complicated by the fact that the method entails culturing of different cell lines with potentially different speciﬁc productivities and growth rates. We have previously published a controlled method for production of rpAb using site-speciﬁc integration technol- ogy [12] to minimize differences in growth rate and productivity from genomic position effects [13]. This technology (named SympressTM I) is being used for the production of an rpAb against the RhD antigen consisting of 25 individual antibodies. This manufacturing strategy has proven to result in highly consistent rpAb compositions from batch-to-batch. The product Sym001 (Rozrolimupab) is presently in clinical phase II. While SympressTM I is well suited for manufacturing of anti-Rhesus D rpAb, where the drug product demands on a yearly basis are relatively small, certain indications with a high product demand (e.g., many cancers) require a higher production level. For this purpose we have tested a number of alternative approaches. The most successful results regarding productivity and batch-to-batch consistency were obtained using a random integration approach. The feasibility of this approach was demonstrated in a new producer cell termed ECHO, a CHO DG44 cell derivative engineered for improved productivity at Symphogen. Using this system we were able to improve productivity signiﬁcantly in a fed batch manufacturing process and at the same time keep the polyclonal compo- sition consistent from batch-to-batch. Materials and Methods Generation of the ECHO Cell Line The mammalian expression cell line used is a derivative of the DHFR-negative Chinese Hamster Ovary (CHO) cell line DG44 kindly provided by Dr. Lawrence Chasin, Columbia University (also available from Gibco cat # 12613-014). The DG44 cells were transfected with a cDNA encoding the 13S version of the adenovirus type 5 Mol Biotechnol (2010) 45:257–266 transactivator E1A (NCBI accession no. AY339865) in the vector pcDNA3.1 ? (Invitrogen). Transfectants were selected with Geneticin (Invitrogen) and single-cell cloned by limiting dilution. A clone showing a transient antibody expression level increased by a factor of 3 compared to the untransfected DG44 cell line was termed the ECHO cell line. Antibody Expression Vector The employed antibody expression vector is shown in Fig. 1a. Antibody heavy and light chain expression is driven by two identical bi-directional human CMV Fig. 1 a Mammalian expression vector used for expression of the anti-Vaccinia and anti-RSV antibodies. CMV human CMV promoters, VH antibody heavy chain variable region, IgG1 constant genomic DNA encoding the human IgG1 constant region, IRES ? DHFR ECMV internal ribosome entry sequence ? mouse DHFR cDNA, SV40 poly A the SV40 polyadenylation sequence, bla and Amp ampicillin resistance gene, pUC ori pUC origin of replication, bGH poly A bovine growth hormone polyadenylation sequence, LC antibody light chain cDNA. b Comparison of speciﬁc productivities in selected pools of DG44 and ECHO cells. DG44 cells and ECHO cells were transfected with three different anti-Vaccinia virus antibody constructs (Ab numbers refer to the numbers used in Fig. 2). Speciﬁc productivities were measured in selected pools