Mol Biotechnol (2010) 45:257–266 DOI 10.1007/s12033-010-9270-9 R E S E A R C H Single-Batch Production of Recombinant Human Polyclonal Antibodies Lars S. Nielsen • Alexandra Baer • Christian Mu¨ ller • Kristian Gregersen • Nina T. Mønster • Søren K. Rasmussen • Dietmar Weilguny • Anne B. Tolstrup Published online: 20 March 2010 The Author(s) 2010. This article is published with open access at Springerlink.com Abstract We have previously described the development and implementation of a strategy for production of recombinant polyclonal antibodies (rpAb) in single batches employing CHO cells generated by site-speciﬁc integra- tion, the SympressTM I technology. The SympressTM I technology is implemented at industrial scale, supporting a phase II clinical development program. Production of recombinant proteins by site-speciﬁc integration, which is based on incorporation of a single copy of the gene of interest, makes the SympressTM I technology best suited to support niche indications. To improve titers while main- taining a cost-efﬁcient, highly reproducible single-batch manufacturing mode, we have evaluated a number of dif- ferent approaches. The most successful results were obtained using random integration in a new producer cell termed ECHO, a CHO DG44 cell derivative engineered for improved productivity at Symphogen. This new expression process is termed the SympressTM II technology. Here we describe proof-of-principle data demonstrating the feasi- bility of the SympressTM II technology for single-batch rpAb manufacturing using two model systems each com- posed of six target-speciﬁc antibodies. The compositional stability and the batch-to-batch reproducibility of rpAb produced by the ECHO cells were at least as good as observed previously using site-speciﬁc integration tech- nology. Furthermore, the new process had a signiﬁcant titer increase. L. S. Nielsen A. Baer C. Mu¨ller K. Gregersen N. T. Mønster S. K. Rasmussen D. Weilguny A. B. Tolstrup (&) Symphogen A/S, Elektrovej, Building 375, DK-2800 Lyngby, Denmark e-mail: abt@symphogen.com Keywords Antibody production Recombinant polyclonal antibodies Clonal cell lines Compositions of cell lines DHFR selection Random integration CHO-DG44 Batch-to-batch consistency Single-batch manufacturing Introduction Plasma-derived immunoglobulins have been used for pas- sive immunotherapy for more than a century [1]. Current use of this type of products includes: treatment of infec- tions with hepatitis B virus, cytomegalovirus, and rabies virus, while plasma-derived immunoglobulin containing anti-Rhesus D antibody is in use for prevention of hemo- lytic disease of the newborn and treatment of idiopathic thrombocytopenic purpura [2–4]. Plasma-derived immu- noglobulins are, however, associated with several disad- vantages such as low efﬁcacy due to a low concentration of antigen-speciﬁc antibodies and potential safety issues due to the risk of disease transmission. Target-speciﬁc recom- binant polyclonal antibodies (rpAb) would potentially overcome these problems [5–7]. Monoclonal antibodies, the second generation of anti- body therapeutics, have been in medical use since the late 1980s. Monoclonal antibodies overcome the above-men- tioned shortfalls of plasma-derived immunoglobulins, but they are less effective against diseases where a complex antigen is the cause [8]. Because monoclonal antibodies bind to only one single structure out of many on the surface of a complex antigen, they are less likely to be able to completely neutralize or eliminate that antigen. Methods for development and manufacture of recom- binant monoclonal antibodies are well established [9–11]. However, a new strategy is required to allow adaptation of