260 Bioreactor Fed Batch Productions For the fed batch productions of the anti-Vaccinia rpAb compositions, one pWCB ampoule was thawed and trans- ferred to ProCHO4 medium in a shaker ﬂask and incubated at 37 C and 5% CO2. The cells were counted three times a week and diluted while expanding the volume as necessary. After 14 days, a 500 ml DASGIP bioreactor was inocu- lated with 0.6 9 106 cells per ml in 250 ml ProCHO4 medium at 80 rpm and 37 C. The culture was fed with ProCHO4 in a growth-rate-dependent manner up to 120 h, after which the feed was decreased gradually. Concentra- tions of glucose and L-glutamine were adjusted throughout, and sodium bicarbonate was used for pH adjustment. IgG product titer was determined by IgG speciﬁc HPLC. The cultivation was continued until the cell viability was below 70% (around 16 days) and the cells were removed from the supernatant by centrifugation and sterile ﬁltration. The fed batch productions of the anti-RSV rpAb com- positions were performed in a similar manner, except that a chemically deﬁned medium was used instead of the ProCHO4 medium and that a chemically deﬁned feed was used instead of ProCHO4. IgG ELISA IgG was measured by sandwich ELISA. Brieﬂy, 96-well plates (Maxisorp, NUNC) were coated with goat anti- human Fc (Serotec, STAR106) followed by incubation with samples and standard (a puriﬁed human monoclonal IgG1 kappa antibody). Detection was performed with goat anti-human kappa light chains conjugated with horseradish peroxidase (Serotec STAR100P). Mol Biotechnol (2010) 45:257–266 with 5–10 ml 0.22 lm ﬁltered medium supernatant, the column was washed with 10 ml PBS pH 7.4 and eluted with 0.1 M glycine, pH 2.7. The eluted product was dia- lyzed twice against 50 mM Na-acetate, 40 mM NaCl, pH 5.0. For the anti-RSV rpAb the same puriﬁcation procedure was applied with a load of approximately 15 ml on a 50 ll self-packed column (Poly-Prep Column, Bio-Rad). The eluted product (250 ll) was adjusted to pH 7.0 using 4.5 ll 1.0 M Tris base. Total IgG concentration was determined by measuring the absorbance at 280 nm. CIEX Chromatography The puriﬁed anti-Vaccinia virus rpAb samples were ana- 2 lyzed by CIEX on a PolyCat A column (4.6 9 100 mm , 3 lm, 1500 A˚ from PolyLC Inc., Columbia, MD). The puriﬁed anti-RSV rpAb samples were analyzed by CIEX on a ProPac WCX-10 column (4 9 250 mm2 from Dio- nex). Both columns were equilibrated in 25 mM sodium acetate, pH 5.0, before application of 60 lg of the IgG mixture with a ﬂow rate of 1 ml/min. The anti-Vaccinia virus antibody peaks were subse- quently eluted with a linear gradient from 150 to 500 mM NaCl in 25 mM sodium acetate, pH 5.0 over 44 CV. The anti-RSV antibody peaks were eluted with a linear gradient from 125 to 350 mM NaCl in 25 mM sodium acetate, pH 5.0, in 10 column volumes. The antibody peaks were detected spectrophotometrically at 215 nm. The chro- matograms were peak integrated and the relative areas of the individual peaks determined. Measurement of IgG Concentration by HPLC Results Cell supernatants were centrifuged at 14,0009g, diluted 1:1 with PBS and sterile ﬁltrated through a Millex-GV SLGV013SL Durapore 0.22 lm ﬁlter. Samples were run 2 on a Poros A column (2.1 9 30 mm , Applied Biosys- tems). 5–1000 ll were injected depending on the IgG concentration. Solvents: A Solvent: 19 PBS, B Solvent: 12 mM HCl; 150 mM NaCl, C Solvent: 20% Acetic Acid; 0.3 M MgCl2. System: Dionex HPLC Summit system with Pump p680—UVD170U detector—ASI-100 automated sample injector with 1000 ll loop. A puriﬁed monoclonal IgG1 kappa antibody was used as standard. Puriﬁcation The anti-Vaccinia virus recombinant polyclonal antibodies were puriﬁed on a 1 ml MabSelect SuRe column (GE Healthcare) pre-equilibrated in PBS, pH 7.4. After loading Derivation of the ECHO Cell Line CHO DG44 cells were transfected with a cDNA for the adenoviral transactivator E1a and transfectants were selected with G418. Clones were screened in a transient transfection assay with an antibody expression construct. One clone showed consistently more than a factor 3 improvement in ﬁnal antibody titer. This clone was sub- cloned twice and termed the ECHO cell line. Transient transfections with CMV-based EGFP constructs similarly showed increased expression in the ECHO cell line. Experiments aiming at identifying E1a transcripts by quantitative PCR in the cell line have been unsuccessful (not shown) so it is not clear whether E1a expression is mediating the higher expression or whether the cell has gained improved expression characteristics by random means during sub-cloning.