Appl Microbiol Biotechnol (2006) 72: 1125–1135 DOI 10.1007/s00253-006-0401-y BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING A. S. Coroadinha . P. M. Alves . S. Sá Santos . P. E. Cruz . O.-W. Merten . M. J. T. Carrondo Retrovirus producer cell line metabolism: implications on viral productivity Received: 9 November 2005 / Revised: 9 February 2006 / Accepted: 3 March 2006 / Published online: 6 April 2006 # Springer-Verlag 2006 Abstract The production of retroviral vectors by human cell lines is still hampered by low titers making it relatively difficult to produce very large quantities of this vector of high interest for clinical gene therapy applications. Thus, to improve vector production, we studied the influence of different sugars alone or combinations of sugars on cell growth, vector titers, and metabolism of the producer cell. The use of fructose at 140 mM or a mixed medium (with glucose at 25 mM and fructose at 140 mM) improved the virus titer three- to fourfold, respectively, and the producer cell productivity by fivefold. The increase in the cell productivity was due to a 1.5-fold increase in the vector stability, the remaining increase being due to higher cell specific productivity. The increase in the productivity was associated with lower glucose oxidation and an increase in the lactate and alanine yield. In the mixed medium, an increase in fatty acids derived from the glucose was observed in parallel with a reduction of glutamate and glutamine synthesis via the tricarboxylic acid (TCA) cycle acetyl-CoA and α-ketoglutarate, respectively. Although the higher productivities were associated with severe changes in the glycolysis, TCA cycle, and glutaminolysis, the cell energetic status monitored by phosphocreatine and adenosine triphosphate levels was not significantly affected. The synthesis of fatty acids and phospholipids were enhanced in the fructose or mixed media and are possibly key parameters in retroviral vector production. Introduction Gene therapy is a promising technology with the potential to correct inherited diseases and treat acquired illness such as cancer or infectious diseases (Andreadis et al. 1999; Mountain 2000; Thomas et al. 2003). The therapy is based on the delivery of genetic material into somatic cells resulting in the correction of the biochemical defects; viral vectors are the vehicles of choice. Retroviral vectors based on Moloney murine leukemia virus (MoMLV) are used in a wide range of clinical trials presenting main advantages such as, the ability to integrate into the genome, maintain a long-term expression, and having a relatively low immu- nogenic toxicity (Mountain 2000; Relph et al. 2004). However, the clinical applications of retroviral vectors for gene therapy are limited by the relatively low productivities of the retroviral packaging cell lines and by the low stability of the vector in culture (Chuck and Palsson 1996; McTaggart and Al-Rubeai 2000). In general, gene therapy applications require gene transfer of a large number of cells demanding high quantities of high titer retroviral prepara- tions (Andreadis et al. 1999; Davis et al. 1997; Dunbar and Kohn 1996; Lyddiatt and O’Sullivan 1998; McTaggart and Al-Rubeai 2000). Therefore, one of the major challenges facing the field of gene therapy is the improvement of the production processes to attain sufficient quantities of retroviral vectors. It was extensively reported in the literature that several culture parameters may affect the vector production yield; these include temperature, pH, serum concentration, me- dium composition, level of inhibitory metabolic by- products, and cell culture density. These studies showed that significant improvements in vector production can be accomplished by simple optimization of some of those A. S. Coroadinha . P. M. Alves . S. S. Santos . P. E. Cruz . M. J. T. Carrondo (*) IBET/ITQB, Apartado 12, Oeiras 2781-901, Portugal e-mail: mjtc@itqb.unl.pt Tel.: +351-214427787 Fax: +351-214421161 P. E. Cruz ECBIO, Lab 4.11, Ed. ITQB, Apartado 98, Oeiras 2781-901, Portugal O.-W. Merten Généthon, 1 bis Rue de l’Internationale, BP 60, Evry, Cedex 91002, France M. J. T. Carrondo Laboratório de Engenharia Bioquímica, FCT/UNL, Monte da Caparica 2825, Portugal