Transcriptome Dynamics of Transgene Amplification in Chinese Hamster Ovary Cells Nandita Vishwanathan, 1 Huong Le, 1 Nitya M. Jacob, 1 Yung-Shyeng Tsao, 2 Sze-Wai Ng, 3 Bernard Loo, 3 Zhong Liu, 2 Anne Kantardjieff, 1 Wei-Shou Hu 1 1 Department of Chemical Engineering and Materials Science, University of Minnesota, 421 Washington Avenue S.E., Minneapolis, Minnesota 55455-0132; telephone: þ1-612-625-0546; fax: þ1-612-626-7246; e-mail: acre@umn.edu 2 Bioprocess Development, Merck & Co., Union, New Jersey 3 Bioprocessing Technology Institute, Singapore, Singapore ABSTRACT: Dihydrofolate reductase (DHFR) system is used to amplify the product gene to multiple copies in Chinese Hamster Ovary (CHO) cells for generating cell lines which produce the recombinant protein at high levels. The physiological changes accompanying the transformation of the non-protein secreting host cells to a high producing cell line is not well characterized. We performed transcriptome analysis on CHO cells undergoing the selection and amplification processes. A host CHO cell line was transfected with a vector containing genes encoding the mouse DHFR (mDHFR) and a recombinant human IgG (hIgG). Clones were isolated following selection and subcloned following amplification. Control cells were transfected with a control plasmid which did not have the hIgG genes. Although methotrexate (MTX) amplification increased the transcript level of the mDHFR gene significantly, its effect on both hIgG heavy and light chain genes was more modest. The subclones appeared to retain the transcriptome signatures of their parental clones, however, their productivity varied among those derived from the same clone. The transcript levels of hIgG transgenes of all subclones fall in a narrower range than the product titer, alluding to the role of many functional attributes, other than transgene transcript, on productivity. We cross examined functional class enrichment during selection and amplification as well as between high and low producers and discerned common features among them. We hypothesize that the role of amplification is not merely increasing transcript levels, but also enriching survivors which have developed the cellular machinery for secreting proteins, leading to an increased frequency of isolating high- producing clones. We put forward the possibility of assembling a hyper-productivity gene set through compara- tive transcriptome analysis of a wide range of samples. Biotechnol. Bioeng. 2014;111: 518–528. ß 2013 Wiley Periodicals, Inc. KEYWORDS: gene amplification; dihydrofolate reductase (DHFR); methotrexate (MTX); selection; hyper- productivity; Chinese Hamster Ovary (CHO) cells; mammalian cell culture Introduction A large number of recombinant protein therapeutics are produced in Chinese Hamster Ovary (CHO) cells (Dean and Reddy, 2013; Jayapal et al., 2007; Kim et al., 2012; Wurm, 2004). In the past quarter century, we have witnessed a two order of magnitude increase in product titer (Wurm, 2004). Critical to attaining a high productivity process is the establishment of a high producing cell line which can secrete the product to a high level in a timely fashion. Many of the high producing CHO cell lines were obtained using the dihydrofolate reductase (DHFR)-based amplifica- tion system developed three decades ago. DHFR catalyzes the conversion of dihydrofolic acid to tetrahydrofolic acid, a precursor for the synthesis of glycine, thymidine phosphate, and purine. CHO cell lines, such as DG44 and DXB11, which are mutated to be deficient in DHFR activity, require supplementation of hypoxanthine and thymidine (HT- media) for growth. Upon co-introduction of the exogenous DHFR gene with the product transgene, cells that express the transgene can be selected in HT-deficient media (Urlaub and Chasin, 1980). By including in the medium, a high level of methotrexate (MTX), a DHFR inhibitor, one can further select for cells which have elevated levels of DHFR. The selected cells typically have multiple copies of an approxi- mately 100 kb region of the chromosomal region containing The present address of Huong Le and Nitya M. Jacob are Amgen, Inc. The present address of Anne Kantardjieff is Alexion Pharma, Cheshire, Connecticut Nandita Vishwanathan, Huong Le, Nitya M. Jacob, and Yung-Shyeng Tsao contributed equally to this work. Correspondence to: W.-S. Hu Contract grant sponsor: Vietnam Education Foundation Contract grant sponsor: NIH Biotechnology Training Grant Contract grant number: GM08347 Received 29 May 2013; Revision received 16 August 2013; Accepted 9 September 2013 Accepted manuscript online 21 September 2013; Article first published online 18 October 2013 in Wiley Online Library (http://onlinelibrary.wiley.com/doi/10.1002/bit.25117/abstract). DOI 10.1002/bit.25117 ARTICLE 518 Biotechnology and Bioengineering, Vol. 111, No. 3, March, 2014 ß 2013 Wiley Periodicals, Inc.