Mol Biotechnol (2010) 45:257–266 promoters. The mouse dihydrofolate reductase (DHFR) is used as selection marker. DHFR expression is coupled to expression of the antibody heavy chain by an encepha- lomyocarditis virus (EMCV) internal ribosome entry site (IRES). Antibodies For initial proof-of-principle experiments six different IgG1 kappa antibodies directed against different Vaccinia virus surface proteins were isolated using the SymplexTM technology [14]. The genes encoding the variable heavy and light chains of the individual antibodies were each cloned into the mammalian cell expression vector shown in Fig. 1a. The antibodies are further described in WO 2007/ 065433. For the second model system, six different respiratory syncytial virus (RSV) speciﬁc IgG1 kappa antibodies iso- lated from infected individuals using the SymplexTM technology and selected for reactivity with the RSV F- and G- surface proteins were used. Stable Transfection of ECHO Cells and Single Cell Cloning by FACS ECHO cells were seeded in T80 ﬂasks at a density of 0.15 9 106 cells/ﬂask in MEM alpha medium with nucleosides (MEMalpha ? medium; Invitrogen) with 10% fetal calf serum (FCS) (Invitrogen). After 24 h the cells were transfected using Fugene6 (Roche) according to the manufacturer’s instructions. Pools of transfectants were selected with 2 nM (for generation of anti-Vaccinia antibody-expressing ECHO cells) or 3 nM (for generation of anti-RSV antibody- expressing ECHO cells) methotrexate (MTX) in nucleo- side-free medium supplied with 10% dialyzed FCS (MEMalpha-medium). The MTX-selected pools were sin- gle-cell cloned by FACS based on high cell surface expression of IgG. Brieﬂy, cells were detached, stained with phycoerythrin-coated goat anti-human IgG (IM1626, Beckman Coulter) and single-cell sorted by gating the upper (with regard to Mean Fluorescence Intensity) 1–2% into 96-well plates containing 100 ll of a 1:1 mix of MEMalpha-medium to conditioned MEMalpha ? medium from non-transfected ECHO cells. No MTX was added to the culture medium from this point. The 96-well plates were visually screened for the presence of single-cell clones and after 10–13 days supernatants were screened by IgG ELISA. ELISA values and visual inspection of the degree of conﬂuence in the wells were used to select clones for further culture. Selected clones were transferred and propagated in T80 ﬂasks and speciﬁc productivities were measured by IgG 259 ELISA. The results were used to select clones for cryo- preservation and adaptation to serum-free suspension culture. Adaptation to Serum-free Suspension Culture in ProCHO4 Adherent cells were adapted to serum-free suspension culture in ProCHO4 medium (ProCHO4 (Lonza), 4 mM L-glutamine (Invitrogen), 1% non-essential amino acids (Invitrogen), anti-clumping agent 1:250 (Invitrogen)) in aerated 50 ml cell culture tubes (TPP). The tubes were placed in a shaker incubator at 37 C and 5% CO2 at a shaking speed of 200 rpm. The medium was changed or the cultures diluted as needed. When growth was vig- orous (doubling time below 30–35 h) the cells were considered adapted, after which they were expanded and frozen. Adaptation to Chemically Deﬁned Medium The anti-RSV ECHO clones in ProCHO4 medium were transferred to shaker ﬂasks in chemically deﬁned medium at a shaking speed of 100 rpm. Medium exchange or dilution of cultures was performed when needed. The adaptation lasted around 3 weeks and was considered complete when doubling times were stable and below 35 h. Co-Cultivation of Six Anti-Vaccinia Clones The anti-Vaccinia co-cultivation experiment was per- formed in aerated 50 ml culture tubes in a total volume of 10 ml. The experiment was started with a cell concentra- tion of 0.3 9 106 cells/ml. The cultures were diluted to 0.3 9 106 cells/ml twice a week with 3 and 4 days inter- vals. Once a week samples for ELISA and cation exchange chromatography (CIEX) analysis were taken starting on day 4 and ending on day 35. Preparation of Polyclonal Cell Banks The two-tiered preparation of polyclonal cells banks is shown schematically in Fig. 4. Brieﬂy, the chosen clones in logarithmic growth phase were mixed, aliquoted, and fro- zen in a number of ampoules (polyclonal master cell bank (pMCB)). Polyclonal working cell banks (pWCB) were prepared by thawing a pMCB ampoule, expanding the cells for a period, aliquoting and freezing a number of pWCB ampoules. The exact culture period at each step and the number of cells frozen differed from experiment to experiment (see ‘‘Results’’ section).