The artificial chromosome expression (ACE) technology system uses an engineered artificial chromosome containing multiple site-specific recombination acceptor sites for the rapid and efficient construction of stable cell lines. The construction of Chinese hamster ovary (CHO) cell lines expressing an IgG1 monoclonal antibody (MAb) using the ACE system has been previously described (Kennard et al., Biotechnol Bioeng. 2009; 104: 540-553). To further demonstrate the manufacturing feasibility of the ACE system, four CHO cell lines expressing the human IgG1 MAb 4A1 were evaluated in batch and fed-batch shake flasks and in a 2-L fed-batch bioreactor. The batch shake flasks achieved titers between 0.7 and 1.1 g/L, whereas the fed-batch shake flask process improved titers to 2.5-3.0 g/L. The lead 4A1 ACE cell line achieved titers of 4.0 g/L with an average specific productivity of 40 pg/ (cell day) when cultured in a nonoptimized 2-L fed-batch bioreactor using a completely chemically defined process. Generational stability characterization of the lead 4A1-expressing cell line demonstrated that the cell line was stable for up to 75 days in culture. Product quality attributes of the 4A1 MAb produced by the ACE system during the stability evaluation period were unchanged and also comparable to existing expression technologies such as the CHO-dhfr system. The results of this evaluation demonstrate that a clonal, stable MAb-expressing CHO cell line can be produced using ACE technology that performs competitively using a chemically defined fed-batch bioreactor process with comparable product quality attributes to cell lines generated by existing technologies. (C) 2010 American Institute of Chemical Engineers Biotechnol. Prog., 27: 201-208, 2011